Glucagon and/or IGF-1 Production Regulates Resetting of the Liver Circadian Clock in Response to a Protein or Amino Acid-only Diet.

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.

Fish Oil Accelerates Diet-Induced Entrainment of the Mouse Peripheral Clock via GPR120.

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock.

Feeding and adrenal entrainment stimuli are both necessary for normal circadian oscillation of peripheral clocks in mice housed under different photoperiods.

The mammalian circadian rhythm is entrained by multiple factors, including the light-dark cycle, the organism's feeding pattern and endocrine hormones such as glucocorticoids. Both a central clock (the suprachiasmatic nucleus, or SCN) and peripheral clocks (i.e. in the liver and lungs) in mice are entrained by photoperiod. However, the factors underlying entrainment signals from the SCN to peripheral clocks are not well known. To elucidate the role of entrainment factors such as corticosterone and feeding, we examined whether peripheral clock rhythms were impaired by adrenalectomy (ADX) and/or feeding of 6 meals per day at equal intervals under short-day, medium-day and long-day photoperiods (SP, MP and LP, respectively). We evaluated the waveform and phase of circadian rhythms in the liver, kidney and salivary gland by in vivo imaging of PER2::LUCIFERASE knock-in mice. In intact mice, the waveforms of the peripheral clocks were similar among all photoperiods. The phases of peripheral clocks were well adjusted by the timing of the "lights-off"-operated evening (E) oscillator but not the "lights-on"-operated morning (M) oscillator. ADX had almost no effect on the rhythmicity and phase of peripheral clocks, regardless of photoperiod. To reduce the feeding-induced signal, we placed mice on a restricted feeding regimen with 6 meals per day (6 meals RF). This caused advances of the peripheral clock phase in LP-housed mice (2-5 h) and MP-housed mice (1-2 h) but not SP-housed mice. Thus, feeding pattern may affect the phase of peripheral clocks, depending on photoperiod. More specifically, ADX + 6 meals RF mice showed impairment of circadian rhythms in the kidney and liver but not in the salivary gland, regardless of photoperiod. However, the impairment of peripheral clocks observed in ADX + 6 meals RF mice was reversed by administration of dexamethasone for 3 days. The phase differences in the salivary gland clock among SP-, MP- and LP-housed mice became very small following treatment with ADX + 6 meals RF, suggesting that the effect of photoperiod was reduced by ADX and 6 meals RF. Because the SCN rhythm (as evaluated by PER2 immunohistochemistry) was not disrupted by ADX + 6 meals RF, impairment of peripheral clocks in these mice was not because of impaired SCN clock function. In addition, locomotor activity rhythm and modifications of the feeding pattern may not be completely responsible for determining the phase of peripheral clocks. Thus, this study demonstrates that the phase of peripheral clocks responds to a photoperiodic lights-off signal, and suggests that signals from normal feeding patterns and the adrenal gland are necessary to maintain the oscillation and phase of peripheral clocks under various photoperiods.

Controlling access time to a high-fat diet during the inactive period protects against obesity in mice.

Free feeding (FF) with a high fat diet (HFD) causes excessive body weight gain, whereas restricted feeding (RF) with a HFD attenuates body weight gain. The effects of timing of feeding with a HFD (day vs. night) and feeding duration on energy homeostasis have not yet been investigated. In this study, we fed mice a HFD or a normal diet (ND) twice a day, during their active and inactive periods, on a schedule. The amount of food was regulated by feeding duration (2, 4 or 8 h). First, we investigated the effects of 4-h RF during active-inactive periods (ND-ND, HFD-HFD, ND-HFD or HFD-ND). Among all the 4-h RF groups, mice consumed almost the same amount of calories as those in the FF[ND] group, even those fed a HFD. Body weight and visceral fat in these three groups were lower than that in the FF[HFD] group. Second, we investigated the effects of RF duration. Body weight and visceral fat were higher in the 8-h groups than in the 4-h groups. Body weight and visceral fat were higher in the 2-h groups than in the 4-h groups even though the 2-h groups had less food. Third, we investigated the effects of eating a HFD during the inactive period, when RF duration was extended (2, 6 or 12 h). Mice were fed with a HFD during the inactive period for 2 h and fed with a ND during the active period for 2, 6 or 12 h. Body weight and visceral fat in these mice were comparable to those in the FF[ND] mice. The results of our first set of experiments suggest that 4-h RF was an adequate feeding duration to control the effect of a HFD on obesity. The results of our second set of experiments suggest 2-h RF (such as speed-eating) and 8-h RF, representative of eating disorders, are unhealthy feeding patterns related to obesity. The results of our third set of experiments suggest that eating a HFD for a short period during the night does not affect body weight and visceral fat. Taken together, these results indicate that consideration to feeding with a HFD during the inactive period and restricting eating habits relieve the risks of body weight gain and visceral fat accumulation.

Combination of meal and exercise timing with a high-fat diet influences energy expenditure and obesity in mice.

In mice, obesity has been observed not only in those freely fed a high-fat diet (HFD) but also in those fed while physically inactive. In contrast, a HFD during physically active periods protects against obesity and the impairments in the circadian rhythm induced by free feeding of a HFD. Although exercise is known to be effective for obesity prevention and management, the optimal timing of exercise has not yet been determined. In the present experiments, we aimed to determine the best combination of daily timing of HFD consumption and exercise for the prevention of HFD-induced weight gain in mice. In this experiment, "morning" refers to the beginning of the active phase (the "morning" for nocturnal animals). Increases in body weight related to free feeding of a HFD was significantly reduced with 4 h of exercise during the late (evening) or middle (noon) active period compared to 4 h of exercise during the early (morning) active period or free access to exercise, which resulted in hours of exercise similar to that of morning exercise. These results suggested that eating in the morning or at noon followed by exercise in the evening could prevent weight gain more effectively than exercise in the morning followed by eating at noon or in the evening. The group fed a HFD for 4 h in the morning had lower body weight than the group fed a HFD for 4 h in the evening without exercise. The last group of experiments tested the hypothesis that there would be an interaction between mealtime and exercise time (i.e. time of day) versus order (i.e. which comes first) effects. We compared groups that exercised for 4 h at noon and were fed either in the morning or evening and groups that were fed for 4 h at noon and either exercised in the morning or evening. We found that the groups that were fed before exercise gained less body and fat weight and more skeletal muscle weight compared to the groups that exercised before eating. Corresponding to the body and fat weight changes, the respiratory exchange ratio (RER) was lower and energy expenditure was higher in the groups fed before exercise than in the groups fed after exercise, and these effects on energy metabolism were also observed in the early stage of HFD feeding before obesity. When obese mice fed a HFD for 12 weeks were exposed to a combination of feeding and exercise timing in an effort to reduce body weight, eating followed by exercise resulted in greater weight loss, similar to the experiments conducted to prevent weight gain. These results demonstrate that a combination of daily timing of eating and exercise may influence weight gain and that eating followed by exercise may be effective for minimizing increases in body and fat weight as well as maximizing increases in skeletal muscle weight.

Time-restricted feeding of rapidly digested starches causes stronger entrainment of the liver clock in PER2::LUCIFERASE knock-in mice.

Restricting feeding to daytime can entrain circadian clocks in peripheral organs of rodents, and nutrients that rapidly increase the blood glucose level are suitable for inducing entrainment. However, dietetic issues, for example, whether or not the diet comprises heated food, have not been fully explored. We therefore hypothesized that rapidly digested starch causes stronger entrainment than slowly digested starch. The entrainment ability of the liver clock in PER2::LUCIFERASE knock-in mice, blood glucose levels, insulin levels, and acute changes in liver clock gene expression were compared between a ß-starch (native)-substituted AIN-93M standard diet and an α-starch (gelatinized)-substituted diet. ß-Corn and ß-rice starch induced larger phase delays of the liver clock, larger blood glucose increases, and higher Per2 gene expression in the liver compared with ß-potato starch. Starch granule size, as examined by electron microscopy, was larger for ß-potato starch than for ß-corn or ß-rice starch. After heating, we obtained gelatinized α-potato, α-corn, and α-rice starch, which showed destruction of the crystal structure and a high level of gelatinization. No difference in the increase of blood glucose or insulin levels was observed between ß-corn and α-corn starch, or between ß-rice and α-rice starch. In contrast, α-potato starch caused higher levels of glucose and insulin compared with ß-potato starch. An α-potato starch-substituted diet induced larger phase delays of the liver clock than did ß-potato starch. Therefore, rapidly digested starch is appropriate for peripheral clock entrainment. Dietetic issues (heated vs unheated) are important when applying basic mouse data to humans.