Effect of circadian clock and claudin regulations on inulin-induced calcium absorption in the mouse intestinal tract

Abstract

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 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 Ca²⁺-free diet, Ca²⁺-containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hr. 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.

Keywords: calcium absorption; circadian rhythm; claudin; clock gene; prebiotics; tight junction.

Fig. 1.

Diurnal variations of clock genes and calcium absorption-related genes in the intestine. Daily mRNA expression rhythms of the core clock genes Per2 (A) and Bmal1 (B) and intestinal calcium absorption-related genes Cldn2 (C), Cldn15 (D), Trpv6 (E), and Pmca1 (F) in the intestine (blue, jejunum; pink, ileum; green, colon) by qPCR. Tissue samples were collected every 4 hr over the course of a day under ad libitum feeding of control AIN-93 M diet. The white and dark bars at the bottom indicate the light and dark periods, respectively. Data are expressed as mean ± SEM values. N=6 at each time point.

Fig. 2.

Elevated Claudin expression levels in the ZT0 inulin-fed group. Day-night changes in Claudin expression levels under time-restricted fiber feeding conditions. The mRNA expression levels of Cldn2 (A, C) and Cldn15 (B, D) in the intestine (upper, jejunum; lower, ileum) were measured for each experimental condition (5% cellulose and 5% inulin diets; blue, ZT0 feeding; orange, ZT12 feeding) by qPCR. Vertical lines through boxes indicate the median. The whiskers go from each quartile to the minimum or maximum. N=5–6 each. **p<0.01, *p<0.05.

Fig. 3.

Inulin intake effectively enhances urine the Ca²⁺/Creatinine ratio in mice fed at ZT0. Time-dependent calcium administration and urine excretion in mice under time-restricted feeding conditions. Urine Ca²⁺/creatinine ratios under each of the experimental conditions, Ca²⁺-free diet (A), 5% cellulose diet (B), and 5% inulin diet (C), as a total of different timepoints (blue, ZT0 feeding; orange, ZT12 feeding). Comparison of urine Ca²⁺/creatinine ratios according to duration after each meal (black, Ca²⁺-free diet; green, 5% cellulose diet; orange, 5% inulin diet) for the different feeding times: ZT0 feeding (D) and ZT12 feeding (E). Vertical lines through boxes indicate the median. The whiskers go from each quartile to the minimum or maximum. Since some mice did not urinate during sampling period, more mice (n=20) were prepared, and the number of collected samples was not the same at each time point (n=5–13 each). ****p<0.0001, **p<0.01, *p<0.05.

Fig. 4.

Effect of SCN lesion on time-dependent Ca²⁺ administration and urine excretion. Day-night variations of the core clock genes Per2 (A) and Bmal1 (B) were detected in the jejunum under each experimental condition (5% cellulose and 5% inulin diets; blue, ZT0 feeding; orange, ZT12 feeding) by qPCR. Urine Ca²⁺/creatinine ratio (C) and cecal pH (D) under each experimental condition. Day-night variations of cldn2 were detected in the jejunum (E) and ileum (F) under each experimental condition (5% cellulose and 5% inulin diets; blue, ZT0 feeding; orange, ZT12 feeding) by qPCR. Vertical lines through boxes indicate the median. The whiskers go from each quartile to the minimum or maximum. N=5–7 each. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.