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* This work was supported in part by Grant-in-Aid (18300228) for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan, and Grant from Fuji Foundation for Protein Research. 1 Present address: Dept. of Biological Science, Faculty of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan.
Sterol regulatory element-binding protein-1 (SREBP-1) plays a central role in transcriptional regulation of genes for hepatic lipid synthesis that utilizes diet-derived nutrients such as carbohydrates and amino acids, and expression of SREBP-1 exhibits daily rhythms with a peak in the nocturnal feeding period under standard housing conditions of mice. Here, we report that the Srebp-1 expression rhythm shows time cue-independent and Clock mutation-sensitive circadian nature, and is synchronized with varied photoperiods apparently through entrainment of locomotor activity and food intake. Fasting caused diminution of Srebp-1 expression, while diabetic db/db and ob/ob mice showed constantly high expression with loss of rhythmicity. Time-restricted feedings during mid-light and mid-dark periods exhibited differential effects, the latter causing more severe damping of the oscillation. Therefore, “when to eat in a day (the light/dark cycle),” rather than “whenever to eat in a day,” is a critical determinant to shape the daily rhythm of Srebp-1 expression. We further found that a high-carbohydrate diet and a high-protein diet, as well as a high-fat diet, cause phase shifts of the oscillation peak into the light period, underlining the importance of “what to eat.” Daily rhythms of SREBP-1 protein levels and Akt phosphorylation levels also exhibited nutrient-responsive changes. Taken together, these findings provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of the Srebp-1 expression and possibly a number of other physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.
The circadian timekeeping system for physiology and behavior in mammals consists of a whole-body network of cell-intrinsic oscillators that rely on activation/repression-alternating feedback loops of clock gene expression (
). SREBP-1a and SREBP-1c are products of the Srebp-1 gene with alternative usage of different 5′ exons 1a and 1c, respectively, while SREBP-2 derives from a separate gene. These isoforms preferentially activate different sets of genes: SREBP-1c, genes for triglyceride synthesis; SREBP-2, genes for cholesterol synthesis; and SREBP-1a, genes both for triglyceride and cholesterol synthesis (
). Precursor forms of SREBPs located to the endoplasmic reticulum membrane are converted to nucleus-targeted transcription factors through vesicle transport into the Golgi apparatus and succeeding two-step proteolytic cleavage, in response to sterol depletion for SREBP-1a and SREBP-2 (
) levels exhibit daily rhythms with a peak during the feeding period, i.e. the dark period in the liver of nocturnal mice fed with standard chows. Here, we at first characterized general features of daily Srebp-1 expression rhythms, which exhibited time cue-independent and Clock mutation-sensitive circadian nature, entrainability to various photoperiods, and fasting- and diabetes-labile damping. We further showed that “when to eat in a day (the light/dark cycle)” under time-restricted feeding conditions and “what to eat” with chows varied in the composition of three major nutrients are principle determinants in shaping the Srebp-1 expression rhythm and possibly a number of other physiological rhythms.
Results of this work led us to hypothesize about mechanisms of daily regulation of Srebp-1 expression, as illustrated in Fig. 9. The schema for ad libitum feeding of the standard diet (Fig. 9A) was drawn from the observations that the Srebp-1 expression rhythm showed circadian nature (Fig. 2) susceptible to Clock mutation (Fig. 3), and that varied photoperiods entrained the Srebp-1 expression rhythm as well as daily rhythms of locomotor activity, food intake and the clock-related gene expression in the liver (Fig. 4). In Fig. 9A, the light-entrainable central clock, which is located in the SCN and adjusted everyday by light/dark cues conveyed from the eye (
). Feeding-associated factors such as nutrients entrain the circadian clock of the liver, in phase with neuronal and hormonal factors under the control of the SCN and the “feeding clock.” All together, nutrients, the liver clock oscillator, and neuronal/hormonal factors in phase cooperate to bring about robustly rhythmic expression of the Srebp-1 gene.
Under time-restricted feeding (Fig. 9B), food intake entrains clocks of most peripheral tissues and even of most brain regions besides the SCN more strongly than the light/dark cues do (
). A typical example of the brain food-entrainable oscillator constituting at least a part of the “feeding clock” is located in the dorsomedial hypothalamic nucleus, which is responsible for food-anticipatory behavior under the restricted feeding (
). The food-entrained clock in the liver regulates Srebp-1 expression in cooperation with almost in-phase factors such as nutrients. On the other hand, SCN-governed relatively out-of-phase factors likely affect also the Srebp-1 expression, as typically remarked under 4-h restricted feeding in the dark period compared with that in the light period (Fig. 6B). Namely, daytime and nighttime feeding differentially affect food-entrainable rhythmicity of Srebp-1 expression. Thus, “when to eat in a day (the light/dark cycle),” rather than “whenever to eat in a day,” is an essential determinant of daily rhythms of Srebp-1 expression. Constantly high-level Srebp-1 expression lacking apparent rhythmicity in db/db and ob/ob mice (Fig. 5) may reflect attenuated feeding rhythmicity accompanied by arrhythmic locomotion (
As for diets with varied nutritional compositions (Fig. 9C), their effects on the systemic daily rhythms such for locomotor activity and feeding may be different from one another, but in any way seem limited (Refs.
), the high-fat diet caused phase advance of the Srebp-1 mRNA rhythm. Furthermore, we found that the high-carbohydrate and high-protein diets also caused phase advance of the Srebp-1 mRNA rhythm with a shifted peak in the light period. Aberrations in daily rhythms of SREBP-1 protein levels were also observed (Fig. 7, bottom panels). Therefore, “what to eat” affects the daily rhythm of Srebp-1 expression profoundly, while its effect on clock-related gene expression is modest. Plausible explanation for these differential effects of varied nutrients is as follows: daily oscillation of clock gene expression in the liver is relatively resistant to nutrient changes, in order to keep time for feeding behavior-associated liver functions that must be entrained to the feeding period; on the other hand, the daily expression rhythm of Srebp-1, one of the most pivotal genes in metabolic regulation, likely requires to be modulated sensitively in response to nutrient variations. Taken together, views in Fig. 9, B and C, provide a model for mechanisms by which time of day and nutrients in feeding shape daily rhythms of a number of physiological functions with interindividual and interdaily differences in human beings and wild animals subjected to day-by-day changes in dietary timing and nutrients.
Interestingly, all three nonstandard diets used in this study caused phase shift of the Srebp-1 expression rhythm. Because SREBP-1 is an activator of genes involved in fatty acid biosynthesis utilizing diet-derived nutrients such as carbohydrates and carbon skeletons of amino acids, the Srebp-1 expression profile with rapid increase in the early dark (feeding) period on the standard diet appears reasonable or “normal,” compared with “abnormal” profiles on the varied diets. We can rationalize that the “standard” diet has been empirically selected referring to animal health conditions such as growth rate, fertility, and longevity. It is tempting to speculate that “standard” diet-driven “normal” daily expression rhythms of metabolism-regulating genes such as Srebp-1 are prerequisite for animal health. On the other hand, further investigation into formation mechanisms and biological meanings of the varied diet-driven “abnormal” profiles will give insights into mechanisms for adaptation of lipogenesis to nutritional changes.
We thank H. Sugiyama and Y. Tamura for advice in statistical analysis, A. Oohira for help in sequence analysis, and members of our laboratories for suggestions, help, and discussions.