The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment.

Soy protein intake is known to cause microbiota changes. While there are some reports about the effect of soy protein intake on gut microbiota and lipid metabolism, effective timing of soy protein intake has not been investigated. In this study, we examined the effect of soy protein intake timing on microbiota. Mice were fed twice a day, in the morning and evening, to compare the effect of soy protein intake in the morning with that in the evening. Mice were divided into three groups: mice fed only casein protein, mice fed soy protein in the morning, and mice fed soy protein in the evening under high-fat diet conditions. They were kept under the experimental condition for two weeks and were sacrificed afterward. We measured cecal pH and collected cecal contents and feces. Short-chain fatty acids (SCFAs) from cecal contents were measured by gas chromatography. The microbiota was analyzed by sequencing 16S rRNA genes from feces. Soy protein intake whether in the morning or evening led to a greater microbiota diversity and a decrease in cecal pH resulting from SCFA production compared to casein intake. In addition, these effects were relatively stronger by morning soy protein intake. Therefore, soy protein intake in the morning may have relatively stronger effects on microbiota than that in the evening.

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Dietary calcium supplementation has been shown to be an effective adjunct therapy in an inflammatory bowel disease model. Soluble dietary fiber reduces intestinal pH and is known to enhance calcium absorption. Although many circadian clock regulations of nutrient absorption in the 2 intestinal tract have been reported, the effects of clock regulation on calcium absorption have yet to be understood. In this study, we investigated the timing of efficient calcium intake by measuring urinary calcium excretion in mice. The diurnal variations in channel-forming tight junctions (claudins) were detected in both the jejunum and ileum. Following 2 days of feeding with a Ca 2+ -free diet, Ca 2+containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hours. There was an evident increase in urinary calcium concentration when the inulin diet was fed at the beginning of the resting period. The Claudin 2 (Cldn2) expression level also showed a significant day-night change, which seemed to be a mechanism for the increased calcium excretion after inulin intake. This diurnal rhythm and enhanced Cldn2 expression were abolished by disruption of the suprachiasmatic nucleus, the central clock in the hypothalamus. This study suggests that intestinal calcium absorption might be modulated by the circadian clock and that the intake of inulin is more effective at the beginning of the resting period in mice.

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