Metabolic adaptations of liver mitochondria during restricted feeding schedules

Food anticipatory activity (FAA) is an output of the food-entrained oscillator (FEO), a conspicuous biological clock that expresses when experimental animals are under a restricted food schedule (RFS). We have shown that the liver is entrained by RFS and exhibits an anticipatory response before meal time in its oxidative and energetic state. The present study was designed to determine the mitochondrial oxidative and phosphorylating capacity in the liver of rats under RFS to further support the biochemical anticipatory role that this organ plays during the food entrainment (9). Metabolic and functional parameters of liver mitochondria were characterized before (0800 h), during (1100 h), and after (1400 h) FAA. The main results were as follows. First, there was an enhancement during FAA (1100 h) in 1) oxidative capacity (site I of the electron transport chain), 2) phosphorylating ability (estimated by ATP synthesis), and 3) activities of NADH shuttles. Second, after rats were fed (1400 h), the phosphorylating capacity remained high, but this was not the case for the respiratory control ratio for site I. Finally, in the three experimental conditions before, during, and after FAA, an increment was detected in the H(+) electrochemical potential, due to an elevation in mitochondrial membrane potential, and in mitochondrial yield. Most of the changes in mitochondrial properties related to RFS were also present when results were compared with those from the 24-h fasted group. In conclusion, the results support the notion that a distinctive rheostatic state is installed in the metabolic activity of the liver when FEO is being expressed.     

RATS MADE CIRRHOTIC BY CHRONIC CCl4 TREATMENT STILL EXHIBIT ANTICIPATORY ACTIVITY TO A RESTRICTED FEEDING SCHEDULE

Food entrainment of clock genes in the liver suggests that this organ may underlie a food entrained oscillator (FEO), which manifests under restricted feeding schedule (RFS). In order to test the importance of a functional liver for the expression of FEO, chronic CCl₄-treated cirrhotic rats and oil-treated controls were entrained to RFS and drinking behavior was continuously monitored. After 20 d of free-running conditions, food access was restricted to 2 h, followed by a refeeding–fasting protocol to test persistence of anticipatory drinking. Present data show no differences between groups for the onset and intensity of anticipation during RFS. After RFS, however, cirrhotic rats exhibited a significantly longer free-running period and a delay and lower intensity of the persistence of activity under fasting conditions. Histology confirmed injury of the liver chronically treated with CCl₄. Present data indicate that a dysfunctional liver due to chronic CCl₄ does not prevent animals from exhibiting anticipatory activity but may promote metabolic derangement of the clock mechanisms of the suprachiasmatic nucleus and the FEO.     

Rats made cirrhotic by chronic CCl4 treatment still exhibit anticipatory activity to a restricted feeding schedule

Food entrainment of clock genes in the liver suggests that this organ may underlie a food entrained oscillator (FEO), which manifests under restricted feeding schedule (RFS). In order to test the importance of a functional liver for the expression of FEO, chronic CCl4-treated cirrhotic rats and oil-treated controls were entrained to RFS and drinking behavior was continuously monitored. After 20 d of free-running conditions, food access was restricted to 2 h, followed by a refeeding-fasting protocol to test persistence of anticipatory drinking. Present data show no differences between groups for the onset and intensity of anticipation during RFS. After RFS, however, cirrhotic rats exhibited a significantly longer free-running period and a delay and lower intensity of the persistence of activity under fasting conditions. Histology confirmed injury of the liver chronically treated with CCl4. Present data indicate that a dysfunctional liver due to chronic CCl4 does not prevent animals from exhibiting anticipatory activity but may promote metabolic derangement of the clock mechanisms of the suprachiasmatic nucleus and the FEO.     

Is the food‐entrainable circadian oscillator in the digestive system?

Food-anticipatory activity (FAA) is the increase in locomotion and core body temperature that precedes a daily scheduled meal. It is driven by a circadian oscillator but is independent of the suprachiasmatic nuclei. Recent results that reveal meal-entrained clock gene expression in rat and mouse peripheral organs raise the intriguing possibility that the digestive system is the site of the feeding-entrained oscillator (FEO) that underlies FAA. We tested this possibility by comparing FAA and Per1 rhythmicity in the digestive system of the Per 1-luciferase transgenic rat. First, rats were entrained to daytime restricted feeding (RF, 10 days), then fed ad libitum (AL, 10 days), then food deprived (FD, 2 days). As expected FAA was evident during RF and disappeared during subsequent AL feeding, but returned at the correct phase during deprivation. The phase of Per1 in liver, stomach and colon shifted from a nocturnal to a diurnal peak during RF, but shifted back to nocturnal phase during the subsequent AL and remained nocturnal during food deprivation periods. Second, rats were entrained to two daily meals at zeitgeber time (ZT) 0400 and ZT 1600. FAA to both meals emerged after about 10 days of dual RF. However, all tissues studied (all five liver lobes, esophagus, antral stomach, body of stomach, colon) showed entrainment consistent with only the night-time meal. These two results are inconsistent with the hypothesis that FAA arises as an output of rhythms in the gastrointestinal (GI) system. The results also highlight an interesting diversity among peripheral oscillators in their ability to entrain to meals and the direction of the phase shift after RF ends.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO's free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO's free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO’s free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO’s free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats

Restricted feeding schedules entrain behavioral and physiological circadian rhythms, which depend on a food-entrainable oscillator (FEO). The mechanism of the FEO might depend on digestive and endocrine processes regulating energy balance. The present study characterizes the dynamics of circulating corticosterone, insulin, and glucagon and regulatory parameters of liver metabolism in rats under restricted feeding schedules. With respect to ad libitum controls, food-restricted rats showed 1) an increase in corticosterone and glucagon and a decrease in insulin before food access, indicating a predominant catabolic state; and 2) a reduction in lactate-to-pyruvate and beta-hydroxybutyrate-to-acetoacetate ratios, indicating an oxidized cytoplasmic and mitochondrial redox state in the liver metabolism. All these changes were reversed after feeding. Moreover, liver energy charge in food-restricted rats did not show a significant modification before feeding, despite an increase in adenine nucleotides, but showed an important decrease after food intake. Variations detected in the liver of food-restricted rats are different from those prevailing under 24-h fasting. These observations suggest "anticipatory activity" of the liver metabolism to optimize the processing of nutrients to daily feeding. Data also suggest a possible relationship of the liver and endocrine signals with the FEO.     

Scheduled feeding alters the timing of the suprachiasmatic nucleus circadian clock in dexras1-deficient mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1(-/-)) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1(-/-) mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1(-/-) mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca ).     

THE FOOD-ENTRAINABLE OSCILLATOR: A NETWORK OF INTERCONNECTED BRAIN STRUCTURES ENTRAINED BY HUMORAL SIGNALS?

Food is critical for all animal species. Its temporal availability is a relevant signal for organizing behavioral and physiological parameters. When food is restricted to a few hours per day, rats, mice, and other mammals exhibit anticipatory activity before mealtime (food-anticipatory activity). There is considerable evidence suggesting that this anticipation is mediated by a food-entrainable oscillator (FEO) with circadian properties, but located outside the suprachiasmatic nucleus of the hypothalamus (the light-entrainable oscillator). However, the locus of the FEO as well as the mechanisms by which food entrainment occurs is unclear. In this review, we summarize data about the potential input pathways to the FEO and propose a model for understanding it as a network of interconnected brain structures entrained by fluctuation of different humoral signals. (Author correspondence: brenotercio@cb.ufrn.br).     

Daily Epinephrine but not Norepinephrine Administration Produces Anticipatory Drinking Behavior in Rats

Peripheral oscillators, especially in the liver, are thought to be part of the food entrained oscillator (FEO). The internal mediating signals involved in food entrainment are unknown. Catecholamines may be one signal, since they increase their blood concentration immediately after feeding, and their intraperitoneal (IP) administration produces hyperglycemia and a satiety response by their direct effect on the liver. The present study explored the role of epinephrine (EPI) and norepinephrine (NE) as entraining signals to the FEO. This effect was explored by daily IP administration of EPI (12.5 µg/100 g bw), NE (25 µg/100 g b) or vehicle on rhythmicity of drinking behavior in rats maintained under constant conditions. Behavioral effects were compared with those observed in a group entrained with a restricted feeding schedule (RFS). IP administration of EPI produced anticipatory drinking behavior, in a dosedependent manner but with a lower intensity than that produced by RFS. In contrast, daily NE or vehicle administration did not produce any changes in drinking rhythmicity. None of the manipulations produced effects on the SCN-dependent freerunning period. Present data suggest a role of EPI as a factor involved with the internal mediating signal for FEO; however, other preabsortive or postabsortive signals may also be part of this complex entraining pathway.     

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.     

Short-term fasting affects locomotor activity,corticosterone, and corticosterone binding globulin in a migratory songbird

Unpredictable events such as severe storms lead to an increase in circulating corticosterone (CORT) in breeding birds. This increase is often accompanied by elevations in foraging and irruptive behavior. We were interested in determining if acute food restriction (such as might occur during inclement weather) is a sufficient cue to elicit an increase in locomotor activity, increase CORT secretion, and/or decrease circulating levels of corticosterone binding globulin (CBG) in white-crowned sparrows (Zonotrichia leucophrys gambelii). Male Z.l. gambelii were housed individually in environmental chambers on long days (LD 20:4) to simulate breeding season daylength. Birds were fed ad libitum, and on select days, food was removed 2 h after lights on (fasted treatment), or was removed and replaced (control). We analyzed CORT and CBG levels after 1, 2, 6, 22 (lights on), and 23 h under fasted and control conditions. We also measured activity during the 23-h experiment. Activity levels were increased under fasted conditions during the daytime relative to control conditions, but activity levels did not differ between treatments during the night. Fasting as little as 1, 2, and 6 h significantly increased total CORT levels above baseline (control), although after 22 h, total CORT levels under fasted conditions matched those under control conditions. Plasma CBG decreased after the 22-h fast, and remained low after the 23-h fast. This change was sufficient to significantly elevate free CORT levels in fasted birds relative to ad libitum food conditions, despite the lack of difference in total CORT levels.     

Isolating neural correlates of the pacemaker for food anticipation

Mice fed a single daily meal at intervals within the circadian range exhibit food anticipatory activity. Previous investigations strongly suggest that this behaviour is regulated by a circadian pacemaker entrained to the timing of fasting/refeeding. The neural correlate(s) of this pacemaker, the food entrainable oscillator (FEO), whether found in a neural network or a single locus, remain unknown. This study used a canonical property of circadian pacemakers, the ability to continue oscillating after removal of the entraining stimulus, to isolate activation within the neural correlates of food entrainable oscillator from all other mechanisms driving food anticipatory activity. It was hypothesized that continued anticipatory activation of central nuclei, after restricted feeding and a return to ad libitum feeding, would elucidate a neural representation of the signaling circuits responsible for the timekeeping component of the food entrainable oscillator. Animals were entrained to a temporally constrained meal then placed back on ad libitum feeding for several days until food anticipatory activity was abolished. Activation of nuclei throughout the brain was quantified using stereological analysis of c-FOS expressing cells and compared against both ad libitum fed and food entrained controls. Several hypothalamic and brainstem nuclei remained activated at the previous time of food anticipation, implicating them in the timekeeping mechanism necessary to track previous meal presentation. This study also provides a proof of concept for an experimental paradigm useful to further investigate the anatomical and molecular substrates of the FEO.     

Scheduled Feeding Alters the Timing of the Suprachiasmatic Nucleus Circadian Clock in Dexras1-Deficient Mice

Restricted feeding (RF) schedules are potent zeitgebers capable of entraining metabolic and hormonal rhythms in peripheral oscillators in anticipation of food. Behaviorally, this manifests in the form of food anticipatory activity (FAA) in the hours preceding food availability. Circadian rhythms of FAA are thought to be controlled by a food-entrainable oscillator (FEO) outside of the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. Although evidence suggests that the FEO and the SCN are capable of interacting functionally under RF conditions, the genetic basis of these interactions remains to be defined. In this study, using dexras1-deficient (dexras1^?/?) mice, the authors examined whether Dexras1, a modulator of multiple inputs to the SCN, plays a role in regulating the effects of RF on activity rhythms and gene expression in the SCN. Daytime RF under 12L:12D or constant darkness (DD) resulted in potentiated (but less stable) FAA expression in dexras1^?/? mice compared with wild-type (WT) controls. Under these conditions, the magnitude and phase of the SCN-driven activity component were greatly perturbed in the mutants. Restoration to ad libitum (AL) feeding revealed a stable phase displacement of the SCN-driven activity component of dexras1^?/? mice by ～2?h in advance of the expected time. RF in the late night/early morning induced a long-lasting increase in the period of the SCN-driven activity component in the mutants but not the WT. At the molecular level, daytime RF advanced the rhythm of PER1, PER2, and pERK expression in the mutant SCN without having any effect in the WT. Collectively, these results indicate that the absence of Dexras1 sensitizes the SCN to perturbations resulting from restricted feeding. (Author correspondence: haiying.cheng@utoronto.ca)     

Persistence of a behavioral food-anticipatory circadian rhythm following dorsomedial hypothalamic ablation in rats

Circadian rhythms of behavior in rodents are regulated by a system of circadian oscillators, including a master light-entrainable pacemaker in the suprachiasmatic nucleus that mediates synchrony to the day-night cycle, and food-entrainable oscillators located elsewhere that generate rhythms of food-anticipatory activity (FAA) synchronized to daily feeding schedules. Despite progress in elucidating neural and molecular mechanisms of circadian oscillators, localization of food-entrainable oscillators driving FAA remains an enduring problem. Recent evidence suggests that the dorsomedial hypothalamic nucleus (DMH) may function as a final common output for behavioral rhythms and may be critical for the expression of FAA (Gooley JJ, Schomer A, and Saper CB. Nat Neurosci 9: 398-407, 2006). To determine whether the reported loss of FAA by DMH lesions is specific to one behavioral measure or generalizes to other measures, rats received large radiofrequency lesions aimed at the DMH and were recorded in cages with movement sensors. Total and partial DMH ablation was associated with a significant attenuation of light-dark-entrained activity rhythms during ad libitum food access, because of a selective reduction in nocturnal activity. When food was restricted to a single 3-h daily meal in the middle of the lights-on period, all DMH and intact rats exhibited significant FAA. The rhythm of FAA persisted during a 48-h food deprivation test and reappeared during a 72-h deprivation test after ad libitum food access. The DMH is not the site of oscillators or entrainment pathways necessary for all manifestations of FAA, but may participate on the output side of this circadian function.     

Circadian rhythm of ornithine decarboxylase activity in small intestine of fasted rats.

The aim of this study was to determine whether the circadian changes in ornithine decarboxylase (ODC) activity of different segments of the small intestine were governed by factors other than food intake. First, the effects of fasting on mucosal ODC activity were examined. The results indicate that mucosal ODC activity in 24 hr and 48 hr fasted rats decreased significantly compared with ad libitum-fed rats. Second, the circadian rhythm of mucosal ODC activity was characterized by measuring mucosal ODC activity in fasted rats at four time points (09:00, 15:00, 21:00, and 03:00 hr; light period: 06:00-18:00 hr). The results from this study indicate that there is a detectable baseline ODC activity in different segments of fasting intestine. In duodenum, mucosal ODC activity was highest at 15:00 hr (light period), a time at which the rat was normally not eating. In jejunum and ileum, mucosal ODC activity increased between 21:00 and 03:00 hr (dark period). The observation that small intestine exhibits a distinct circadian rhythm of ODC activity in fasted rats suggests that not only food but also intrinsic factors can modulate physiologic oscillations in mucosal ODC activity.