Effects of Dietary Protein Intake Levels on Peripheral Circadian Rhythm in Mice.

The regulation of the circadian clock plays an important role in influencing physiological conditions. While it is reported that the timing and quantity of energy intake impact circadian regulation, the underlying mechanisms remain unclear. This study investigated the impact of dietary protein intake on peripheral clocks. Firstly, transcriptomic analysis was conducted to investigate molecular targets of low-protein intake. Secondly, mPer2::Luc knock-in mice, fed with either a low-protein, normal, or high-protein diet for 6 weeks, were analyzed for the oscillation of PER2 expression in peripheral tissues and for the expression profiles of circadian and metabolic genes. Lastly, the candidate pathway identified by the in vivo analysis was validated using AML12 cells. As a result, using transcriptomic analysis, we found that the low-protein diet hardly altered the circadian rhythm in the central clock. In animal experiments, expression levels and period lengths of PER2 were different in peripheral tissues depending on dietary protein intake; moreover, mRNA levels of clock-controlled genes and endoplasmic reticulum (ER) stress genes were affected by dietary protein intake. Induction of ER stress in AML12 cells caused an increased amplitude of Clock and Bmal1 and an advanced peak phase of Per2. This result shows that the intake of different dietary protein ratios causes an alteration of the circadian rhythm, especially in the peripheral clock of mice. Dietary protein intake modifies the oscillation of ER stress genes, which may play key roles in the regulation of the circadian clock.

Anti-obesity effect of butyrate links to modulation of gut microbiome and epigenetic regulation of muscular circadian clock.

The role of the muscle circadian clock in regulating oxidative metabolism exerts a significant influence on whole-body energy metabolism; however, research on the connection between the muscle circadian clock and obesity is limited. Moreover, there is a lack of studies demonstrating the regulatory effects of dietary butyrate on muscle circadian clock and the resulting antiobesity effects. This study aimed to investigate the impacts of dietary butyrate on metabolic and microbiome alterations and muscle circadian clock in a diet-induced obesity model. Male Sprague-Dawley rats were fed a high-fat diet with or without butyrate. Gut microbiota and serum metabolome were analyzed, and molecular changes were examined using tissues and a cell line. Further correlation analysis was performed on butyrate-induced results. Butyrate supplementation reduced weight gain, even with increased food intake. Gut microbiome analysis revealed an increased abundance of Firmicutes in butyrate group. Serum metabolite profile in butyrate group exhibited reduced amino acid and increased fatty acid content. Muscle circadian clock genes were upregulated, resulting in increased transcription of fatty acid oxidation-related genes. In myoblast cells, butyrate also enhanced pan-histone acetylation via histone deacetylase inhibition, particularly modulating acetylation at the promoter of circadian clock genes. Correlation analysis revealed potential links between Firmicutes phylum, including certain genera within it, and butyrate-induced molecular changes in muscle as well as phenotypic alterations. The butyrate-driven effects on diet-induced obesity were associated with alterations in gut microbiota and a muscle-specific increase in histone acetylation, leading to the transcriptional activation of circadian clock genes and their controlled genes.

Effect of aging on the formation and growth of colonic epithelial organoids by changes in cell cycle arrest through TGF-ß-Smad3 signaling.

BACKGROUND: This study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism. METHODS: To investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6-10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups. RESULTS: The number and size of organoids from old mice was significantly lower than that from young mice (p < 0.0001) at day 5 of passage 0. The growth rate of old-mouse organoids from day 4 to 5 of passage 0 was significantly slower than that of young-mouse organoids (2.21 times vs. 1.16 times, p < 0.001). RNA sequencing showed that TGF-ß- and cell cycle-associated genes were associated with the aging effect. With regard to mRNA and protein levels, Smad3 and p-Smad3 in the old-mouse organoids were markedly increased compared with those in the young-mouse organoids. Decreased expression of ID1, increased expression of p16INK4a, and increased cell cycle arrest were observed in the old mouse-organoids. Treatment with SB431542, a type I TGF-ß receptor inhibitor, significantly increased the formation and growth of old-mouse organoids, and TGF-ß1 treatment markedly suppressed the formation of young-mouse organoids. In the acute dextran sulfate sodium-colitis model and its organoid experiments, the colonic epithelial regeneration after tissue injury in old mice was significantly decreased compared with young mice. CONCLUSIONS: Aging reduced the formation ability and growth rate of colonic epithelial organoids by increasing cell cycle arrest through TGF-ß-Smad3-p16INK4a signaling.

Effects of Dietary Fat to Carbohydrate Ratio on Obesity Risk Depending on Genotypes of Circadian Genes.

Although the impacts of macronutrients and the circadian clock on obesity have been reported, the interactions between macronutrient distribution and circadian genes are unclear. The aim of this study was to explore macronutrient intake patterns in the Korean population and associations between the patterns and circadian gene variants and obesity. After applying the criteria, 5343 subjects (51.6% male, mean age 49.4 ± 7.3 years) from the Korean Genome and Epidemiology Study data and nine variants in seven circadian genes were analyzed. We defined macronutrient intake patterns by tertiles of the fat to carbohydrate ratio (FC). The very low FC (VLFC) was associated with a higher risk of obesity than the optimal FC (OFC). After stratification by the genotypes of nine variants, the obesity risk according to the patterns differed by the variants. In the female VLFC, the major homozygous allele of CLOCK rs11932595 and CRY1 rs3741892 had a higher abdominal obesity risk than those in the OFC. The GG genotype of PER2 rs2304672 in the VLFC showed greater risks for obesity and abdominal obesity. In conclusion, these findings suggest that macronutrient intake patterns were associated with obesity susceptibility, and the associations were different depending on the circadian clock genotypes of the CLOCK, PER2, and CRY1 loci.