1Instituto de Neurobiología, Universidad Nacional Autónoma de México-Juriquilla, Querétaro; and 2Facultad de Medicina and 3Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México
Submitted 27 October 2004 ; accepted in final form 13 June 2005
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ABSTRACT |
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metabolism; food-entrainable oscillator; oxidation; swelling; H+ electrochemical potential; change in pH; mitochondrial membrane potential
The FEO is expressed when animals are exposed to a restricted feeding schedule (RFS) of 24 h of food access per day during 2 or 3 wk. This condition increases locomotive activity and arousal during the previous hours of mealtime (26), generating a condition known as food anticipatory activity (FAA). FAA is characterized by physiological and behavioral changes in the organism such as the increment of wheel running activity, water drinking, and body temperature as well as a peak of serum corticosterone (9, 11, 26).
The nature and location of the FEO are still unknown. The working hypothesis of our group is that the FEO is a distributed system constituted by 1) central nervous structures such as hypothalamic nuclei with the capacity to act in response to signals derived from the nutritional state and energetic metabolism and 2) peripheral organs such as the liver, pancreas, or adipose tissue, in which synthesis, oxidation, and coordinated handling of energy metabolites such as carbohydrates, lipids, and amino acids take place. The time-keeping mechanism of the FEO would emerge based in the feedback loops between these two systems by either neuronal and/or humoral signals (2, 11). A crucial feature of this model is the role played by the liver, because this organ is directly involved with food processing, nutrient delivery, and bioenergetic status of the organism (24).
The liver is a peripheral oscillator (36), and reports based on monitoring rhythms of clock genes have indicated that it plays a relevant role in the physiology of FEO: 1) Per1 gene variations in the liver are entrained by feeding (33) and 2) the rhythms of Per1 and other clock genes in hepatic tissue are uncoupled from the SCN by RFS (6). However, some reports dispute the participation of the liver as a direct or principal component of the FEO: 1) peak Per1 in the liver and other digestive organs did not remain in the phase promoted by restricted feeding but shifted back to the nocturnal phase during subsequent periods of ad libitum feeding and food deprivation (8) and 2) cirrhotic rats with hepatic malfunction by chronic CCl4 treatment still exhibited FAA to a RFS (12).
In food-restricted rats, we have reported a correlation between metabolic parameters in the liver concomitant with the expression of FAA (9). The metabolic pattern found in the food-restricted rats presented obvious differences from the patterns displayed by ad libitum-fed rats and 24-h fasted control groups, suggesting that the hepatic physiology during the expression of FEO adopts a novel regulatory status promoted by the RFS. We have found that during FAA, there is a significant reduction of glycogen levels (11), the occurrence of an oxidized cytoplasmic and mitochondrial redox state, an enhancement of ATP and ADP levels (9), and a diminution in activity of the triiodothyronine-forming enzyme 5'-deiodinase (leading to a transient hypothyroidal state in the liver) (1). We also previously found that during food presentation (3 h), the stomach of food-restricted rats was not completely empty and showed the lowest weight along the day (25). In addition, at the onset of FAA, the energetic status switched to a metabolic condition with enhanced lipid mobilization from adipose tissue and an increase in fatty acid hepatic metabolism (11, 25).
With the purpose of gaining more understanding about the energy metabolic adaptations in the liver during food entrainment, the aim of the present study was to characterize in rats under RFS the following mitochondrial properties: 1) levels of mitochondrial protein, 2) the oxidative capacity for substrates to site I and II of the respiratory chain, 3) ATP-generating ability, 4) activities of the aspartate-malate and -glycerophosphate shuttles, 5) components of mitochondrial membrane potential (
), and 6) mitochondrial swelling. The results obtained reinforced the notion that hepatic energy metabolism adopts a new regulatory state when the FEO is being expressed.
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MATERIALS AND METHODS |
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Experimental design. Rats were randomly assigned to one of three groups: 1) control rats fed ad libitum during 3 wk, 2) rats exposed to a daily RFS with access to food from 1200 to 1400 h during 3 wk, and 3) rats fasted for 24 h (killed at 1100 h). At the end of the third week, rats from the ad libitum-fed and RFS groups were killed following the protocol reported by Davidson and Stephan (7) at three different time points: 0800 h (before FAA), 1100 h (during FAA) and 1400 h (after being fed). The 24-h fasted group was killed at 1100 h. Each group contained 6 individuals/time point.
Hepatic mitochondrial fraction.
The liver was removed (3 g) and immediately placed in an ice-cold isolation medium and homogenized [1:10 (wt/vol)]. The isolation medium contained 250 mM sucrose, 2 mM HEPES, 10 mM Tris·HCl, 0.1% fatty acid-free BSA, and 0.5 mM EGTA (pH 7.4). The liver mitochondria fraction was obtained according to a modification of the method of Dumas et al. (10). The liver was homogenized with a Teflon-on-glass homogenizer from Potter-Elvehjem (40 rpm for 10 s). The homogenized tissue was first centrifuged at 500 g for 10 min (in a Sorvall SS-34 centrifuge). The resulting supernatant was newly centrifuged at 8,500 g for 15 min. In this and the following steps, the foamy layer at the top of the supernatant was removed to prevent mitochondrial uncoupling. The pellet was resuspended in the isolation medium without EGTA [1:10 (wt/vol)] with a fine camel hair paint brush and finally centrifuged at 9,500 g for 15 min. This part was repeated two times. The final pellet was resuspended in 0.5 ml of isolation medium (without EGTA). Mitochondrial protein was measured by the Biuret method (14).
Mitochondrial marker enzymes. Citrate synthase (EC 2.3.3.1 [EC] ) was assayed and quantified as described by Venditti et al. (35). Mitochondrial yield was calculated according to the method described by Idell-Wenger et al. (18).
Oximetry. Mitochondrial respiratory activity and oxidative phosphorylation were recorded polarographically with a Clark-type oxygen electrode in 3 ml of respiratory medium as reported by Hernández-Muñoz et al. (17). The incubation medium contained 225 mM sucrose, 10 mM KCl, 5 mM MgCl2, 10 mM KH2PO4, 10 mM Tris·HCl, and 0.1% BSA (pH 7.4). For site I of the electron transport chain (ETC), 10 mM glutamate and 1 mM malate were used as substrates, whereas 5 mM succinate was used for site II of the ETC. Mitochondrial state 3 respiration was stimulated with the addition of ADP (250 µM final concentration), whereas state 4 was evaluated when ADP had been totally consumed. The highest velocity of the ETC (uncoupled state) was promoted by the addition of 2,4-dinitrophenol (DNP; 0.1 mM final concentration). State 3 (plus ADP), state 4 (without ADP), and uncoupled state (plus DNP) were determined as in Hernández-Muñoz et al. (17). Respiratory control was calculated from the state 3-to-state 4 ratio. The phosphorylation capacity of the system was evaluated by the ADP-to-O ratio (ADP/O): 750 nmol ATP formed/total O2 consumed in state 3. ATP synthesis rates were calculated from the product of state 3 respiration x ADP/O for site II substrates.
Mitochondrial shuttles.
The "endogenous" and "reconstituted" activities of the malate-aspartate and -glycerophosphate shuttles were determined using the assay reported by Cederbaum et al. (5), with the modifications used by Hernández-Muñoz et al. (16). Mitochondrial shuttle activities were measured by the disappearance of NADH followed spectrophotometrically at 340 nm. Mitochondria (510 mg) were incubated in a 3-ml final volume of respiratory medium (as described above) in the presence of 5 mM NADH and 5 mM ADP (final concentration). In these conditions, the endogenous activity of the malate-aspartate and the
-glycerophosphate shuttles [with the addition of 12 µM rotenone (final concentration)] were monitored. In the reconstituted experiments, cytosolic components of each shuttle were added to reach maximum activities. The malate-aspartate shuttle was reconstituted with 2 mM aspartate, 5 mM glutamate, 1 mM malate, 25 units malate dehydrogenase, and 2 units glutamic-oxalacetic transaminase, whereas the
-glycerophosphate shuttle was reconstituted with 10 mM
-glycerophsphate, 1 mM ATP, and 3 units
-glycerophosphate dehydrogenase. All mixtures were incubated for 20 min at 37°C, and the reactions were stopped with the addition of 3 M perchloric acid solution (0.2 ml for every 1 ml of mitochondrial suspension) and cooled with ice. The samples were prepared, and shuttle activities were determined according to Klingenberg (22) by quantifying the amount of NAD+ formed by the alcohol dehydrogenase reaction.
Mitochondrial membrane potential.
Mitochondrial was measured by monitoring the movements of [14C]tetraphenylphosphonium (TPP2+) across the mitochondrial membranes in a medium (1.9 ml/sample) containing 40 mM sucrose, 40 mM KCl, 0.4 mM EDTA, 10 mM MgCl2, 50 mM Tris·HCl, 4.8 mM monobasic potassium phosphate (pH 7.4), 21 mM ADP, 100 mM succinate, and 30 µM radiolabeled TPP2+ (30 mCi/mmol specific activity) [Kamo et al. (20), modified by Hernández-Muñoz et al. (17)]. The assay started with the addition of 2 mg mitochondrial protein and lasted 30 min, with the temperature kept at 37°C. After the reaction was stopped by placing the samples on ice, mitochondria were rapidly separated from the medium by centrifugation at 13,000 rpm for 1 min (Eppendorf Centrifuge 5415 C). The supernatants were taken and stored. The mitochondrial pellet was washed twice with the medium described above. The radioactivity contained in the supernatant and pellet was counted after the addition of 10 ml Tritosol.
(distribution of TPP2+) was calculated according to the following Nernst equation:
= RT/nF log (Ci/Ce) (27), where R is the gas constant, T is temperature, n is the charge number of the ion, F is Faraday's constant, Ci is concentration of TPP2+ in mitochondrial matrix, and Ce is concentration of TPP2+ in mitochondrial intermembrane space.
Mitochondrial pH difference.
The difference of pH (pH) across the inner mitochondrial membrane was determined by the equilibrium distribution of radiolabeled [14C]sodium acetate (30 mCi/mmol specific activity) according to Valcarce et al. (24). The technique is similar to the one described for
determination, with the exception that the respiratory medium (see description in Mitochondrial membrane potential) was used during the essay. The
pH (distribution of [14C]acetate) was calculated also by the Nernst equation, as described for
.
Mitochondrial proton-motive electrochemical force.
The value for proton-motive electrochemical force (µH+) was derived from the following equation:
µH+ =
+ 2.303(RT) x
pH/F, where RT is room temperature (27).
Mitochondrial swelling. Mitochondrial swelling was determined according to the method of Packer (29), following the modifications reported by Hernández-Muñoz et al. (17). Changes of mitochondrial volume induced for osmotic modifications with succinate were monitored at 546 nm. Mitochondria (1 mg/ml) were suspended in 10 mM Tris buffer containing 50 mM sucrose, 5 mM MgCl2, 10 mM KCl, and 250 µM ADP (pH 7.5). The reaction was started with the addition of succinate (5 mM final concentration). Difference between initial and final readings at 546 nm after 5 min of recording was representative of swelling activity.
Calculations and statistics.
Results are expressed as means ± SE of at least six individual experimental observations. The statistical significance among RFS and ad libitum-fed groups was assessed with two-way ANOVA (feeding condition x time), followed by a Tukey multiple-comparison post hoc test with set at 0.05. Differences among the RFS group at 1100 h, the ad libitum-fed group at 1100 h, and the 24-h fasted group were assessed with one-way ANOVA (feeding condition), followed by a Tukey multiple-comparison post hoc test with
set at 0.05.
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RESULTS |
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Mitochondrial shuttle activity.
The mitochondrial shuttles are a set of metabolic reactions that allow the introduction of NADH molecules from the cytoplasm into the mitochondria (Fig. 1). Two shuttles are important to introduce NADH into the mitochondria: one that handles malate and aspartate and another that uses -glycerophosphate. Each of them can be studied in a basal or reconstituted state. Figure 3 depicts modifications in the activities of both mitochondrial shuttles associated with RFS. No differences were observed in the ad libitum-fed group in the basal and reconstituted malate-aspartate shuttle (Fig. 3, A and B). Similarly, no changes in the malate-aspartate shuttle were observed between the 24-h fasted group and ad libitum-fed group of rats at 1100 h. In contrast, the RFS group presented significant enhancements in the basal and reconstituted activities of the malate-aspartate shuttle (Fig. 3, A and B). At 0800 h (before FAA), there was a significant augmentation (20%) in the endogenous and reconstituted activities of both mitochondrial shuttles. Major changes were observed again at 1100 h (during FAA) and 1400 h (after rats had been fed) with an increase in the NADH oxidation of more than 300% (endogenous) and 200% (reconstituted), respectively.
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These results further corroborate the enhanced oxidative capacity of liver mitochondria induced by the RFS, specially in the time when FAA is expressed and after mealtime.
Mitochondrial membrane potential.
Transfer of reducing equivalents from NADH or FADH2 to molecular oxygen is coupled to the pumping of protons across the inner mitochondrial membrane. This transport of protons generates µ, which has two components: 1) a pH gradient resulting in
pH across the inner membrane of
1.4 and 2)
, due to the charge separation, equal to about 175 mV (15). Figure 4 shows the modifications in hepatic mitochondrial
pH promoted by the RFS. In the ad libitum-fed group,
did not change in any of the three time points tested (Table 3). The 24-h fasted group did not show any modifications in
. In contrast, the RFS group presented an increase of
40% in the three explored time points. In relation to the ad libitum-fed group,
pH presented the same pattern as
and the 24-h fasted group (Table 3). In the RFS group,
pH showed a significant increase (15%) at 1100 h, coincident with FAA. Figure 4 shows that the pattern displayed by
µ is very similar to the one presented for
(Table 3) in all the experimental groups tested. No significant changes were observed in the ad libitum-fed rats, and there was no effect in the 24-h fasted rats, but there was a
29% augmentation in the RFS group in the three time points studied. The higher
µ observed in the RFS group did not correlate with fasting (0800 and 1100 h) or fed (1400 h) conditions, and it seems to be a characteristic associated with the RFS condition.
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Mitochondrial swelling. Mitochondria present osmotic responses associated with physiological and pathological conditions (37). The osmotic responses can be energy linked or energy independent, and, in some cases, they can be considered as signs of disturbances of the mitochondrial membrane (17). Mitochondrial swelling did not show any modification within the three experimental time points in the ad libitum-fed and RFS groups. By the same token, no changes were detected in the comparison of these experimental groups. The only significant difference observed in this set of data was an increase in the rats with 24-h fasting of 40% and 15% in relation to the ad libitum-fed and RFS groups, respectively (Fig. 5).
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DISCUSSION |
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The mitochondrial fraction recovered from all experimental conditions presented a constant recuperation rate (1819%) and suggested that, although feeding and fasting conditions altered some functional parameters measured in this work, this result presented a homogenized mitochondrial fraction due to the isolation method. To probe whether the RFS condition influenced that some of the mitochondrial subpopulation was more abundant than another or more easily recovered, we needed to apply a methodology to study the properties of all the subpopulations.
Liver mitochondria activity in food-restricted rats.
The data obtained in this study support a direct role of hepatic mitochondria in the augmentation of ATP levels, previously measured by HPLC, and coincident with FAA (9). At this time (1100 h), hepatic mitochondria from the RFS group presented a significant increment in the response to ADP (state 3, site I), corroborated by polarographically oxymetry and an enhanced mitochondrial capacity of phosphorylation (Fig. 2 and Table 2). This higher capacity for ATP synthesis was accompanied by more effective respiratory control with oxidation of substrates for site I of the ETC (Fig. 2). In addition to the enhanced substrate utilization, liver mitochondria during FAA present elevated oxidative power for reducing equivalents, as shown by the higher activity of NADH shuttle systems (Fig. 3). One piece of evidence that RFS produced a new biochemical status in liver mitochondria was the persistent increase in mitochondrial , and hence
µ, in the three experimental time points considered in this study (Table 2). Among the parameters that increase mitochondrial
(19), and compatible with metabolic adaptations present in the liver during expression of the FEO, are 1) a high ATP-to-ADP ratio, 2) dephosphorylation of cytochrome c oxidase at subunit I, 3) the presence of 3,5-diiodo-L-thyronine, and 4) high levels of palmitate. Hyperthyroidism induces an enhancement in the energetic status of mitochondria by increasing the ADP/ATP ratio and by stimulating lipid catabolism in the liver (13). More experiments are needed to define the exact mechanism responsible for the increase in liver mitochondrial
in our experimental protocol. Taken together, these results are a sign of activation in the oxidative and phosphorylative activities of liver mitochondria in rats under RFS. Remarkably, the activation of mitochondrial metabolism takes place before mealtime, when FAA is vigorous, and confirms the notion of "biochemical anticipation" of hepatic physiology before feeding, consistent with the glucogen decrease and enhancement of lipid catabolism and mobilization described by Díaz-Muñoz et al. (9).
Food entrainment promotes rheostatic adaptation in the liver. The whole set of physiological, endocrine, and biochemical changes that have been reported in a variety of tissues in organisms under food entrainment (32) and are highly suggestive of rheostatic adaptation. Rheostasis, formally homeorhesis, was originally defined as the orchestrated changes for priorities of a physiological state (4). Rheostatic control involves the coordination of metabolism, resulting in the direct portioning of nutrient utilization for physiological processes such as lactation, pregnancy, and growth (4). Thus rheostatic mechanisms provide regulation during changes of state, whereas homeostatic control operates on an acute minute-by-minute basis to maintain steadiness of the steady state.
RFS forced experimental animals to ingest in a small interval of 2 h the food that they usually eat in longer periods. This situation requires behavioral, physiological, and metabolic adjustments to permit the organisms to succeed. For this reason, and with a rheostatic perspective, it could be postulated that during food entrainment, there is a shift in the use of nutrients in support of a new physiological state and the implementation of mechanisms by which nutrient handling and partitioning are modified with the ultimate aim to reach a new energetic steady state.
It was pertinent in our experimental protocol to include a 24-h fasted control, because the RFS rats spent 22 h without food. Therefore, RFS rats alternate between a brief state of very intense food ingestion and a larger state of total food deprivation. As a matter of fact, rats under RFS showed a metabolic and physiological pattern that was very different from that present in the ad libitum-fed and 24-h fasted groups (1, 9, 25). One of the key points to explain these dissimilarities is the extreme hyperphagia developed in the 2-h interval in which the RFS rats consume food.
The great hyperphagia measured in rats under RFS represents an adaptative process of the stomach that makes it possible to store large quantities of food and provide uninterrupted nutrient supply to the organism despite the 22-h fasting interval (25). The emptying of the stomach only 3 h before food access (in our experimental protocol, at 1100 h) is coincident with a modification in the bioenergetics of the liver, because it is a change from the handling of sugars to lipids as hepatic metabolic fuels and indicates an adaptive event that suggests that the stomach can serve as a storage site (11, 25). Previously, the stomach presented only slow emptying. The decrease in liver glycogen and the increase of circulating free fatty acids and ketone bodies are concurrent with an augmentation of peroxisomal and mitochondrial catabolic activities for lipids (3). These physiological and metabolic adaptations, acting for days and weeks, could operate in conjunction to induce the new rheostatic handling of nutrients by the liver.
It is widely accepted that behavioral, physiological, and biochemical adaptations induced by RFS reflect a FEO independent of the SCN. The anatomic location of the FEO is still uncertain. So far, the more accepted hypothesis is the one that postulates the FEO as an emerging system based in the metabolic and/or neural communication between peripheral organs and selective brain areas (32).
Independent of the controversy about the precise relevance of the role that the liver plays in the physiology of the FEO (8, 12, 26, 32), it is out of question that food entrainment depends on the availability of energy metabolites and that food processing is largely influenced by the metabolic activity of the liver. Some of the findings reported by our group regarding hepatic metabolic adaptations during food entrainment, such as the increase of ATP, enhancement of acetoacetate (indicating an oxidized redox state), and modulation in the hepatic thyroideal status, point to a direct role played by liver mitochondria in the liver physiology of food-restricted rats (1, 9).
In conclusion, the present results provide evidence that liver mitochondrial activity is modulated by RFS, showing enhanced oxidative and phosphorylative capacity especially during the onset of FAA. The data also strengthen the anticipatory role of the liver as the principal organ in charge of the handling and distribution of nutrients in the 2-h interval of food availability. A model remains to be postulated in which all the biochemical, physiological, and endocrinological modifications attributed to RFS induce the onset of the FEO.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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