Research articleClock genes regulate the feeding schedule-dependent diurnal rhythm changes in hexose transporter gene expressions through the binding of BMAL1 to the promoter/enhancer and transcribed regions
Introduction
Intestinal expression of genes related to hexose absorption in rodents exhibit diurnal periodicity with respect to feeding. Glucose uptake activity in rats fed ad libitum peaks late in the dark phase, or early in the light phase, and depends on the imposed feeding schedule rather than the light cycle [1], [2], [3]. As the food consumption pattern in rats fed ad libitum is established by the light cycle, the diurnal rhythmicity of glucose transport activity is “cued by feeding” rather than as the result of an inherent oscillatory behavior of the small intestine. Expressions of three hexose transporter genes: the sodium/glucose cotransporter (SGLT1) and glucose transporter type 5 (GLUT5), which transport hexose to enterocytes from the lumen, and glucose transporter 2 (GLUT2), which is a glucose/fructose/galactose transporter expressed in the basal membrane in enterocyte, peaks at the late light phase to early dark phase just prior to maximal glucose uptake. In addition, protein levels of SGLT1 and GLUT5, but less so for GLUT2, are associated with expression changes of these genes [4], [5], [6]. The expression of these genes is regulated by the imposed feeding schedule, because feeding rats only during the light phase shifts the peak of expression from late light-early dark phase to late dark-early light phase [3], [7]. These results suggest that the diurnal rhythm of expression of intestinal hexose transporter genes is regulated by feeding rather than light, allowing adaptation to the greater amount of carbohydrate that needs to be digested and absorbed in the small intestine during the dark phase.
Recent studies have demonstrated that diurnal rhythms of gene expression were regulated by CLOCK and BMAL1 proteins, which are heterodimeric nuclear transcription factors. The heterodimer stimulates transcription of “negative regulators” [period 1, 2, and 3 (Per1–3) and cryptochrome 1 and 2 (Cry1–2)] by binding positive cis-elements, called E-boxes, located on their promoter/enhancer regions [8], [9]. The protein products of these genes (Per1–3, Cry1–2) in turn oligomerize, enter the nucleus and suppress the activity of the CLOCK-BMAL1 heterodimer. Additionally, the transcription factor D site albumin promoter binding protein (DBP), is a positive-regulator for Per and Cry expressions, and E4BP4 is a negative-regulator of them. This negative feedback-loop is called the “core-feedback loop of clock genes” [10], [11]. The nuclear receptor RAR-related orphan receptor (ROR) enhances BMAL1, CLOCK and E4BP4 gene expressions; on the other hand, Rev-erb is known to repress their expressions. This feedback mechanism for regulating BMAL1, CLOCK and E4BP4 genes expression is known as the “sub-loop of clock genes” [12], [13]. Recent studies have shown that these feedback loops coordinately regulate diurnal rhythmical expression in the suprachiasmatic nucleus, which is known to regulate the central clock [14].
Recent studies also demonstrated that these clock genes are expressed rhythmically in peripheral organs in which they can presumably coordinate expression of a subset of tissue-specific genes, which might, in turn, impact directly on their physiological functions [9]. Although the central clock in the suprachiasmatic nucleus is light responsive and can be synchronized or reset by environmental cues, such as the light/dark cycle, peripheral clocks are unable to perceive light. These might be entrained by the central clock or independently by other physiological stimuli, such as feeding. Recent studies have demonstrated that feeding is one of the factors that regulate the diurnal rhythm of clock genes in the liver and colon [14]. However, it is not known whether expression of hexose transporters in the small intestine is regulated by these feedback loops of clock genes.
Based on the observation that the gastrointestinal tract is subjected to various 24-h rhythmic processes, we hypothesized that clock genes regulate expression of hexose transporter genes in the small intestine of the mouse. Our results suggest not only that diurnal changes in expression of hexose transporter genes depend on feeding schedule, but also that the expression of the hexose genes is directly regulated by the feedback loop of clock genes.
Section snippets
Animals
Seven-week-old male C57BL/6J mice (Japan SLC, Hamamatsu, Japan) were maintained under a light–dark cycle with 12 h of light and 12 h of darkness per day (light on 07:00, light off 19:00). Animals were divided into two groups. One group had free access to a standard laboratory diet (MF, Oriental Yeast, Tokyo, Japan) for 2 weeks. The other group was subjected to a restricted feeding schedule; they had free access to the laboratory diet only for 8 h during the light period (9:00-17:00). The
Diurnal rhythm of gene expressions for hexose transporters in the jejunum of mice fed ad libitum
To explore whether intestinal genes for hexose transporters demonstrate diurnal rhythms in their gene expressions, we performed real-time RT-PCR using total RNA from the jejunum of the mice fed ad libitum. The expression of hexose transporter genes (SGLT1, GLUT5, and GLUT2) increased from basal levels at 7:00 to maximal levels at 19:00, decreasing thereafter until 3:00 (Fig. 1A).
Diurnal rhythm of expressions for clock genes in the jejunum of the mice fed ad libitum
Next, to explore whether clock genes are expressed and whether they exhibit diurnal rhythm in the small intestine of
Discussion
Hexose transporters (SGLT1, GLUT5, GLUT2) show a diurnal rhythm in their expression in the small intestine of rodents [5], [6]. In this study, we confirmed the results in mice (Fig. 1A). Additionally, we confirmed that the diurnal rhythm is regulated by the imposed feeding schedule (Fig. 2A). Recent studies suggest that the diurnal rhythm of gene expression is regulated by a feedback loop of transcriptional factors and their repressors, called “clock genes.” The feedback loop of clock genes
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (20590233), the Naito Foundation and the Global COE program, Center of Excellence for Innovation of Human Health Sciences, from the Ministry of Education, Science, Sports and Culture of Japan.
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