Comparison of the Effects of Inappropriate Meal Timing-Induced and Genetic Models of Circadian Clock Disruption on Uterine mRNA Expression Profiles

Abstract

Background: Accumulating evidence reveals that inappropriate meal timing contributes to the development of lifestyle-related diseases. An underlying mechanism is thought to be the disruption of the intracellular circadian clock in various tissues based on observations in both systemic and tissue-specific clock gene-deficient mice. However, whether the effects of conditional clock gene knockout are comparable to those of inappropriate meal timing remains unclear.

Objectives: This study aimed to compare the effects of a recently developed 28-h feeding cycle model with those of a core clock gene Bmal1 uterine conditional knockout (Bmal1 cKO) model on uterine mRNA expression profiles.

Methods: The models were generated by subjecting C57BL/6J mice to an 8-h/20-h feeding/fasting cycle for 2 wk and crossing Bmal1-floxed mice with PR-Cre mice. Microarray analyses were conducted using uterine samples obtained at the beginning of the dark and light periods.

Results: The analyses identified 516 and 346, significantly 4-fold and 2-fold, up- or downregulated genes in the 28-h feeding cycle and Bmal1 cKO groups, respectively, compared with each control group. Among these genes, only 7 (1.4%) and 63 (18.2%) were significantly up- or downregulated in the other model. Moreover, most (n = 44, 62.9%) of these genes were oppositely regulated. These findings were confirmed by gene set enrichment analyses.

Conclusions: This study reveals that a 28-h feeding cycle and Bmal1 cKO differently affect gene expression profiles and highlights the need for considering this difference to assess the pathophysiology of diseases associated with inappropriate meal timing.

Keywords: circadian clock; clock genes; gene expression profiles; irregular eating habits; timing of eating; uterus.

FIGURE 1

Heatmap showing the expression profiles of clock genes. Uterine samples were obtained from 10-wk-old C57BL/6J mice (A, C) housed on ad libitum feeding or a 28-h feeding cycle for 2 wk (n = 3 in each group) and Bmal1-floxed mice (B, D) with or without PR-Cre transgene (n = 4 in each group) at ZT 12 (A, B) and 0 (C, D). Red and blue indicate higher and lower expression levels, respectively, compared with each control group. ∗P < 0.05, ∗∗P < 0.01 versus each control group. cKO, conditional knockout; ZT, Zeitgeber time.

FIGURE 2

Heatmap showing the expression profiles of upregulated genes in mice on a 28-h feeding cycle at ZT 12. (A) Significantly upregulated genes with a |log₂ (fold change)| > 2 in the 28-h feeding cycle group, compared with the ad libitum fed control group, at ZT 12 are displayed. (B) Among the genes shown in Figure A, significantly up- or downregulated genes with a |log₂ (fold change)| > 0 in the Bmal1 cKO group, compared with the floxed group, at ZT 12 are displayed. cKO, conditional knockout; ZT, Zeitgeber time.

FIGURE 3

Summary of the effects on gene expression. (A) 28-h feeding cycle model. (B) Bmal1 cKO model. The upper and lower graphs show the data of the up- and downregulated genes, respectively. The left- and right-side graphs demonstrate the data obtained at ZT 12 and 0, respectively. Pie charts and numbers indicate the portions and numbers of the upregulated (green components), downregulated (yellow components), or unchanged genes (light blue components) in the other model. cKO, conditional knockout; FC, fold change; ZT, Zeitgeber time.

FIGURE 4

Heatmap showing the expression profiles of the upregulated genes in Bmal1 cKO mice at ZT 12. (A) Significantly upregulated genes with a |log₂ (fold change)| >1 in Bmal1 cKO mice, compared with the floxed mice, at ZT 12 are displayed. (B) Among the genes shown in Figure A, significantly up- or downregulated genes with a |log₂ (fold change)| > 0 in the 28-h feeding cycle group, compared with the ad libitum fed control group, at ZT 12 are displayed. cKO, conditional knockout; ZT, Zeitgeber time.

FIGURE 5

Hallmark gene sets that were significantly enriched in the 2 models at ZT 12. (A) 28-h feeding cycle model. (B) Bmal1 cKO model. The gene ratio is assigned to the horizontal axis as the proportion of differential genes in the whole gene set. The dot size and color represent the gene number and the P value of each gene set. cKO, conditional knockout; ZT, Zeitgeber time.

FIGURE 6

Hallmark gene sets that were significantly enriched in the 2 models at ZT 0. (A) 28-h feeding cycle model. (B) Bmal1 cKO model. cKO, conditional knockout; ZT, Zeitgeber time.