METABOLIC ADAPTATIONS OF LIVER MITOCHONDRIA DURING

RESTRICTED FEEDING SCHEDULES

Adrián Báez-Ruiz1, Carolina Escobar2, Raúl Aguilar-Roblero3, Olivia Vázquez-Martínez1 and Mauricio Díaz-Muñoz1* 1Instituto de Neurobiología, Campus UNAM-Juriquilla, Apdo. postal 1-1141, Querétaro 76001. 2Facultad de Medicina and 3Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. MÉXICO

ADDRESS FOR CORRESPONDENCE:

Mauricio Díaz Muñoz, Ph.D. Departamento de Neurobiología Celular y Molecular Instituto de Neurobiología, UNAM-Juriquilla Apartado Postal 1-1141 Querétaro, Qro. 76001 México E-mail: mdiaz@calli.inb.unam.mx Telephone and Fax: (5255) 5623-4035

Running Head: liver mitochondria and restricted feeding

Keywords: Liver mitochondria, metabolism, restricted feeding, food entrainable oscillator, oxidation, swelling, ∆Ψ, ∆pH, ∆µ.

Number of figures: 5 Number of tables: 5

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (June 23, 2005). doi:10.1152/ajpgi.00488.2004

Copyright © 2005 by the American Physiological Society.

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ABSTRACT

Food anticipatory activity (FAA) is an output of the food–entrained oscillator (FEO), a

conspicuous biological clock which expresses when experimental animals are under a restricted

food schedule (RFS). We have shown that the liver is entrained by RFS and exhibits anticipatory

response prior to 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 (Díaz-Muñoz et al. 2000). Metabolic and functional parameters of liver mitochondria

were characterized before (08:00 h), during (11:00 h) and after (14:00 h) FAA. The main results

were: A) An enhancement during the FAA (11:00 h) in: 1) oxidative capacity (Site I of the

electron transport chain), 2) phosphorylating ability (estimated by ATP synthesis), 3) activities of

NADH-shuttles; B) After feeding (14:00 h), the phosphorylating capacity remained high but it

was not the case for the respiratory control ratio for site I, and C) In the three experimental

conditions, before, during and after the FAA, an increment was detected in mitochondrial yield

and in the +H-electrochemical potential (∆µ), the latter due to an elevation in mitochondrial

membrane potential (∆Ψ). Most of the changes in mitochondrial properties related to RFS, were

also present when comparing to a 24 h fasting group. In conclusion, the results support the notion

that a distinctive rheostatic state is installed in the metabolic activity of the liver when the FEO is

being expressed.

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INTRODUCTION

Biological rhythms are entrained by cyclic environmental external signals (zeitgebers).

The light-dark cycle is the most consistent environmental 24 h-synchronizer, and its information

is transmitted to the suprachiasmatic nuclei (SCN) of the hypothalamus. The SCN is considered

the master clock of the organism (Klein et al., 1991). Additionally, other recurring inputs, such as

the cyclic access to food, may also act as an entraining factor, by means of a different circadian

oscillator termed the food-entrained oscillator (FEO) (Stephan, 2002).

The FEO is expressed when animals are exposed to a food-restricted schedule (RFS) of 2

to 4 h of food access per day during 2 or 3 weeks. This condition increases locomotive activity

and arousal during the previous hours of mealtime (Mistlberger, 1994), generating a condition

known as food anticipatory activity (FAA). FAA is characterized by physiological and behavioral

changes in the organism such as: increment of wheel running activity, water drinking and body

temperature, as well as a peak of serum corticosterone (Mistlberger, 1994; Escobar et al., 1998

and Díaz-Muñoz et al., 2000).

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 of signals derived from the nutritional

state and energetic metabolism, and 2) Peripheral organs such as 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 (Escobar et al., 1998; Ángeles-Catellanos et al., 2004). A crucial feature of this

model is the role played by the liver, since this organ is directly involved with food processing,

4

nutrients delivery and bioenergetic status of the organism (Langhans, 1996).

Liver is a peripheral oscillator (Yamazaki et al., 2000), and reports based in monitoring

rhythms of clock genes have indicated that plays a relevant role in the FEO’s physiology: 1) Per1

gene variations in the liver are entrained by feeding (Stokkan et al., 2001); 2) The rhythms of Per

1 and other clock genes in hepatic tissue are uncoupled from the SCN by restricted feeding

schedules (Damiola et al., 2000). However, some reports dispute the participation of the liver as a

direct or principal component of the FEO: 1) Peak of Per1 in liver and other digestive organs did

not remain in the phase promoted by restricted feeding, but shifted back to nocturnal phase

during subsequent periods of ad-libitum feeding and food deprivation (Davidson et al., 2003); 2)

Cirrhotic rats with hepatic malfunction by chronic CCl4 treatment still exhibit FAA to a restricted

feeding schedule (Escobar et al., 2002).

In food-restricted rats, we have reported correlation between metabolic parameters in the

liver concomitant with the expression of FAA (Díaz-Muñoz et al., 2000). The metabolic pattern

found in the food-restricted rats presented obvious differences from the patterns displayed by fed

ad-libitum rats and 24 h fasting 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 a significant reduction of glycogen levels (Escobar et al., 1998), the occurrence of an

oxidized cytoplasmic and mitochondrial redox state, an enhancement of ATP and ADP levels

(Díaz-Muñoz et al., 2000), a diminution in activity of the tri-iodothyronine-forming enzyme, 5’-

deiodinase, leading to an transient hypothyroidal state in the liver (Aceves et al., 2003). In

addition, at the onset of FAA the energetic status switched to a metabolic condition with an

enhanced lipid mobilization from adipose tissue, and an increase in fatty acids hepatic

metabolism (Martínez-Merlos et al., 2004 and Escobar et al., 1998). A striking finding involved

in this protocol, was the marked hyperphagia presented by the rats under the food restricted

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schedule, since they had a ≈25 g meal during the 2 h of food accessibility (Martínez-Merlos, et

al., 2004). As a consequence, the full stomach gradually processed the food, becoming empty in a

time coincident with the FAA, the decrease in hepatic glycogen and the enhanced lipid

mobilization (Martínez-Merlos, et al., 2004).

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 next mitochondrial properties: 1) levels of mitochondrial protein; 2) Oxidative capacity

for substrates to site 1 and 2 of the respiratory chain; 3) ATP-generating ability; 4) Activities of

aspartate-malate and α-glycerophosphate shuttles; 5) Components of mitochondrial membrane

potential and 6) Mitochondrial swelling. The results obtained reinforced the notion that the

hepatic energy metabolism adopts a new regulatory state when the FEO is being expressed.

MATERIALS AND METHODS

Animals and Housing

Adult male Wistar rats weighing 180 + 20 g at the beginning of the experiments were

maintained under constant conditions of 12:12-h light-dark cycle (LD, lights on 08:00 h) and

temperature (22 + 1°C). Rats were kept in groups of six in transparent acrylic cages (40 x 5 0 x

20 cm), with free access to Purina Chow and water except during food restriction or fasting

conditions. All experimental procedures were conducted in accordance with our Institutional

Guide for Care and Use of Animal Experimentation (Universidad Nacional Autónoma de

México).

Experimental Design

Rats were randomly assigned to one of three groups: 1) control rats fed ad libitum during

three weeks; 2) rats exposed to a daily restricted feeding schedule (RFS) with access to food from

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12:00 to 14:00 h during three weeks, and 3) rats fasted for 24 h (sacrificed at 11:00 h). At the end

of the third week, rats from ad libitum and RFS groups were sacrificed following the protocol

reported by Davidson et al., 1999, at three different time points: 08:00 h (before FAA), 11:00 h

(during FAA) and 14:00 h (after feeding). The 24 h fasting group, was sacrificed at 11:00 h. Each

group contained 6 individuals per time point.

Hepatic Mitochondrial Fraction

Liver was removed (≈3 g) and immediately placed in an ice-cold isolation medium and

homogenized (1:10 w/v). The isolation medium contained: 250 mM sucrose, 2 mM HEPES, 10

mM Tris/HCl, 0.1% fatty acid-free bovine serum albumin (BSA) and 0.5 mM EGTA (pH 7.4).

Liver mitochondria fraction was obtained according to a modification of the method of Dumas, et

al (2004). Liver was homogenized with a teflon-on-glass homogenizer Potter-Elvehjem (40 RPM

for 10 s). The homogenized tissue was first centrifuged at 500 g x 10 min (in a Sorvall SS-34

centrifuge). The resulting supernatant was newly centrifuged at 8,500 g x 15 min. In this and next

steps, the foamy layer at the top of the supernatant was removed to prevent mitochondrial

uncoupling. The pellet was resuspended in the isolation medium but omitting EGTA (1:10 w/v)

with a fine camel-hair paint brush and finally centrifuged at 9,500 g x 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 (Gornall, 1949).

Mitochondrial Marker Enzymes

Citrate synthase (EC 2.3.3.1) was assayed and quantified as described by Venditti, et al

(2004). Mitochondrial yield was calculated according to the method described by Idell-Wenger,

et al (1982).

Oximetry

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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 (1992). The incubation medium contained 225 mM sucrose, 10 mM

KCl, 5 mM MgCl2, 10 mM KH2PO4, 10 mM Tris-HCl and BSA 0.1% (pH 7.4). For site I of the

electron transport respiratory chain, 10 mM Glutamate (Glu) and 1 mM Malate (Mal) were used

as substrates whereas 5 mM Succinate (Suc) was used for site II. The mitochondrial state 3 was

stimulated adding ADP (250 µM final concentration), whereas state 4 was evaluated when ADP

had been totally consumed. The highest velocity of the electron transport chain (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 (1992). Respiratory control (RC) was calculated from the state 3/state 4

ratio. The phosphorylation capacity of the system was evaluated by the ADP/O ratio: 750 nmols

of 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 α-

glycerophosphate shuttles were determined using the essay reported by Cederbaum et al (19731),

with the modifications used by Hernández-Muñoz et al (1987). Mitochondrial shuttle activities

were measured by the disappearance of NADH followed spectrophotometrically at 340 nm.

Mitochondria (5-10 mg) were incubated in 3 ml of 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 α-glycerophsphate 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

8

malate-aspartate shuttle was reconstituted with 2 mM aspartate, 5 mM glutamate, 1 mM malate,

25 units of malate dehydrogenase and 2 units of glutamic-oxalacetic transaminase; whereas the α-

glycerophosphate shuttle was reconstituted with 10 mM α-glycerophsphate, 1 mM ATP and 3

units of α-glycerophosphate dehydrogenase. All mixtures were incubated for 20 min at 37 °C and

the reactions were stopped with the addition of 3 M of perchloric acid solution (0.2 ml for every

1 ml of mitochondrial suspension) and cooled with ice. The samples were prepared and the

shuttles activities determined according to Klingenberg (1965) by quantifying the amount of

NAD+ formed by the alcohol dehydrogenase reaction.

Mitochondrial ∆Ψ

The mitochondrial membrane potential (∆Ψ) was measured by monitoring the movements

of [14C]tetraphenylphosphonium (TPP2+) across the mitochondrial membranes, in a medium (1.9

ml per sample) containing 40 mM sucrose, 40 mM KCl, 0.4 mM EDTA, 10 mM MgCl2, 50 mM

Tris-HCl, and 4.8 mM monobasic potassium phosphate (pH 7.4), 21 mm ADP, 100 mm succinate

and 30 µM radiolabeled TPP+2 (30 mCi/mmol specific activity) (Kamo et al, 1979, modified by

Hernández-Muñoz, et al, 1992). The essay started with the addition of 2 mg of mitochondrial

protein, and lasted 30 min keeping the temperature at 37 °C. After stopping the reaction by

placing the samples on ice, mitochondria were rapidly separated from the medium by

centrifugation at 13,000 rpm x 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 the pellet was counted after adding 10 ml of

Tritosol. The ∆Ψ (distribution of TPP2+) was calculated according to the Nernst equation: ∆Ψ = -

RT/nF log (Ci/Ce) (Nicholls, 1982).

Mitochondrial ∆pH

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The difference of 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. (1988). The technique is similar to the one described for ∆Ψ

determination, with the exception that the respiratory medium (see description above) was used

during the essay. The ∆pH (distribution of [14C]acetate) was calculated also by the Nernst

equation, as described for ∆Ψ.

Mitochondrial ∆µH+

The value for the proton-motive electrochemical force (∆µH+) was derived from the

following equation: ∆µH+ = ∆Ψ + 2.303RT ∆pH /F, where RT means room temperature and F is

the Faradays constant (Nicholls, 1982).

Mitochondrial Swelling

Mitochondrial swelling was determined according to the method of Packer (1967), and

following the modifications reported by Hernández-Muñoz, et al. (1992). 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

The results are expressed as mean + SEM of at least six individual experimental

observations. The statistical significance among RFS and ad lib groups was assessed with a two-

way ANOVA (feeding condition X time), followed by a Tukey multiple-comparisons post-hoc

test with α set at 0.05. The differences among RFS at 11:00 h, ad- lib at 11:00 h and fasted

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groups were assessed with a one-way ANOVA (feeding condition), followed by a Tukey

multiple-comparisons post-hoc test with α set at 0.05.

RESULTS

A scheme of the main mitochondrial metabolic activities is presented in Figure 1.

Mitochondria are a key factor in the bioenergetic balance of the cell since most of ATP synthesis

occurs in this organelle. Oxidative phosphorylation takes place on its inner membrane whereas

the major catabolic pathways for acetyl coenzyme A (acetyl CoA) production such as pyruvate

degradation and the β-oxidation of fatty acids occur in the mitochondrial matrix. Acetyl CoA

feeds the Krebs cycle to produce reducing equivalents (NADH and FADH2) to activate the

electron transport chain (ETC). The flow of electrons from the reducing equivalents through the

ETC towards O2 promotes the extrusion of H+ from the matrix to the intermembranal space

creating a membrane potential (∆µ). The ∆µ, that includes electrical (∆ψ) and chemical (∆pH)

components, is used by the ATP synthetase to form ATP. NADH produced in the cytosol has

access to the mitochondrial matrix by means of the malate-aspartate and the α-glycerophosphate

shuttles. In addition to oxidative phosphorylation, mitochondria are involved in thermogenesis,

cell death, radical production and modulation of calcium homeostasis (Scheffler 2001).

Mitochondrial Fraction. The use of a marker enzyme of mitochondrial matrix (citrate

synthase activity) and the quantification of protein in homogenate and mitochondrial fraction,

were used to calculate the mitochondrial yield (expressed as mg of protein per liver gram) and the

recovery of this fraction (calculating the recover percentage from the total mitochondrial content

for liver gram) The percentage of mitochondrial protein purified in our experiments ranged from

18%-19% in all the experimental conditions. No significant differences were found in FRS and

control groups (fed ad-libitum and 24 h fasting) in the total mitochondrial content (ratio between

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enzyme activity from homogenate and mitochondrial sample) (Table 1). However, a strong

tendency to present more protein in both, homogenates and mitochondrial fractions of the three

RFS groups, was consistently observed. As a consequence, a significant increase of

mitochondrial yield was observed in the RFS groups in all the three time points studied (average

14% augmentation, Table 1). The higher yield of the mitochondrial fraction in the FRS

presenting FAA (11:00 h), was even more accentuated when it was comparing to the 24 h fasting

rats (24% augmentation). No significant change was observed in mitochondrial yield between ad

libitum fed and 24 h fasted rats (Table 1). These results are indicative that FRS induces in the

liver the presence of more cellular constituents, and hence promotes a higher mitochondrial yield

but without affecting the recovering of this fraction.

Mitochondrial Substrate Oxidation. No significant changes in any of the parameters

associated with the mitochondrial oxidation of glutamate-malate (Site I) and succinate (Site II)

were seen in the three time points in the control group fed ad libitum (Figure 2, Table II). No

changes were either detected when the 24 h fasting control group was compared with the rats fed

ad libitum. Substrates oxidation for Site II, did not present any changes in all experimental

conditions (Figure 2, panel B). In contrast, the RFS group exhibiting FAA (sacrificed at 11:00 h),

presented striking modifications. In comparison to the ad libitum control rats, the RFS group at

11:00 h showed a significant increase (27%) in the respiratory control with substrates of Site I

(Figure 2, Panel A), largely attributable to an elevation of oxygen consumption in the

phosphorylating condition of the state 3. In this group, the ATP synthesis and the maximal

velocity of oxidation promoted by DNP were also enhanced (Table II). The synthesis of ATP

increased 136% for Site I and 167% for Site II. Substrate oxidation in presence of DNP increased

22% for Site I and II among FRS-ad libitum groups at 11:00 h and 36% among FRS and fasted

24 h group. ADP/O values did not change in any experimental condition.

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Taken together, these results indicate a higher oxidative capacity of the liver mitochondria

coincident with the FAA, and support the finding previously reported by our group of an

enhancement in hepatic ATP levels previous to food presentation in RFS (Díaz-Muñoz et al.,

2000).

Mitochondrial Shuttles Activity. The mitochondrial shuttles are a set of metabolic

reactions that allow the introduction of NADH molecules from the cytoplasm into the

mitochondria (Figure 1). Two shuttles are important to introduce NADH into the mitochondria:

one that handles malate and aspartate, and another that uses α-glycerophosphate. Each of them

can be studied with the studied in a basal or reconstituted state. Figure 4 depicts the modifications

in the activities of both mitochondrial shuttles associated to RFS. No differences were observed

in the group fed ad libitum in the basal and reconstituted malate-aspartate shuttle (Figure 3, Panel

A and B). Similarly, no changes in the malate-aspartate shuttle were observed between the 24 h

fasting group and the ad libitum group of rats at 11:00 h. In contrast, RFS group presented

significant enhancements in the basal and reconstituted activities of the malate-aspartate shuttle

(Panel A and B). At 08:00 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 11:00 h (during the FAA) and 14:00 h (after feeding), with an increase in the

NADH oxidation of more than 300% (endogenous) and 200% (reconstituted), respectively.

The profile of activation in the endogenous and reconstituted malate-aspartate shuttle

induced by the RFS, was also observed in the α-glycerophosphate shuttle (Panel C and D).

These results further corroborate the enhanced oxidative capacity of the liver

mitochondria induced by the RFS, specially in the time when FAA is expressed, and after the

meal time.

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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 an electrochemical gradient (∆µ) that has two

components: 1) A pH gradient resulting in a pH difference (∆pH) across de inner membrane of

approximately 1.4, and 2) a membrane potential (∆Ψ), due to the charge separation, of about 175

mV (Heales et al., 2002). Figure 4 shows the modifications in hepatic mitochondrial ∆pH

promoted by RFS. In the group fed ad libitum ∆Ψ did not change in any of the three time points

tested (Table III). Fasting of 24 h did not produced modifications in ∆Ψ. In contrast, the RFS

group presented an increase of ≈40% in the three explored time points. In relation to the group

fed ad libitum, ∆pH presented the same pattern as ∆Ψ and the 24 h fasting group (Table III). In

the RFS group, ∆pH showed a significant increase (15%) at 11:00 h, coincident with the FAA.

Figure 4 shows that the pattern displayed by ∆µ is very similar to the one presented for ∆Ψ in all

the experimental groups tested (Table III). No significant changes are observed in the ad libitum

fed rats, and no effect of the 24 h fasting, but there was a ≈29% augmentation in the RFS groups

in the three times studied. The higher ∆µ observed in the RFS group did not correlate with the

fasting (08:00 and 11:00 h) or fed (14:00 h) conditions, and it seems to be a characteristic

associated with the RFS condition.

Taken together, the data are suggestive of significant modifications in the functioning of

the oxidative phosphorylation in the liver mitochondria of rats under RFS and expressing the

FEO.

Mitochondrial Swelling. Mitochondria present osmotic responses associated with

physiological and pathological conditions (Zoratti and Szabo, 1995). The osmotic responses can

be energy-linked or energy-independent, and in some cases they can be considered as signs of

14

disturbances of the mitochondrial membrane (Hernández-Muñoz et al., 1992). Mitochondrial

swelling did not show any modification within the three experimental points in the group fed ad

libitum and in the RFS group. In the same token, no changes were detected in the comparison

within 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 with the ad libitum and RFS

groups, respectively (Figure 5).

The two-way ANOVA analysis applied in the comparison of RFS and ad-libitum groups,

indicated significant effects of feeding conditions, time points, and their interaction for

mitochondrial yield, respiratory control for site I and II, reconstituted activity of malate-aspartate

and α-glycerophosphate shuttles and ∆µH+. For swelling, the significant effect was detected only

for time point (Table IV). The same results, with the exception of the effect in swelling, were

obtained with the one-way ANOVA analysis applied for the 24 h fasting group. In this case, the

significant changes were detected exclusively when the comparison was done between the 24 h

fasting and the RFS (at 11:00 h) groups (Table IV).

DISCUSSION

In this study, we examined the effect of food restriction and a 24 h fast on the biochemical

properties of liver mitochondria, one of the most influential parameters in the bioenergetic status

of the cell (Heales et al., 2002). Our results indicate that the mitochondrial yield, calculated per

gram of liver, was consistently higher when the experimental animals were under restricted

feeding schedules (Table I). The higher mitochondrial yields were associated with a tendency to

present elevated protein content in liver homogenates in all the three temporal points in which the

food-restricted rats were studied. An interpretation of these results should consider if the water

15

content of the liver could decrease during the feeding restricted schedules. Indeed, we have seen

that rats entrained by feeding present a lower water content in the liver during the FAA (data not

shown, manuscript in preparation). Cellular fractionation to obtain a mitochondrial-enriched

fraction usually involve a recovery of ≈20% (Hernández-Muñoz et al., 1992; Lanni et al., 1996).

Prudence should be taken into account since mitochondrial compartment is highly dynamic and

heterogeneous, i.e., several sub-population of liver mitochondria showing different respiratory,

enzymatic and swelling properties have been reported by Lanni et al., 1996). In the present

experiments, we can not rule out the possibility that food restricted schedules could influence the

kind of mitochondrial sub-population obtained after liver fractionation. The exploration of this

interesting possibility is beyond the aim of this manuscript.

Strengthening the notion of biochemical adaptations of liver mitochondria during the

expression of the OSA, several genes related with mitochondrial physiology were detected to be

up-regulated by DNA microarrays experiments in the livers of rats presenting FAA: the subunits

I, II, III and IV of the cytochrome oxidase complex (COX), diverse subunits of the F0/F1 proton

ATPase and the surfeit locus protein 1 that is localized in mitochondrial inner membrane and is

involved in the cytochrome oxidase complex assemblage (Báez-Ruiz et al., 2005).

Liver Mitochondria Activity in Food-Restricted Rats. 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 the FAA (Díaz-Muñoz et al., 2000). At this time (11:00 h), hepatic

mitochondria from RFS group presented a significant increment in the response to ADP (State 3,

Site I), corroborating by polarographically oxymetry, an enhanced mitochondrial capacity of

phosphorylation (Figure 2, Table II). This higher capacity for ATP synthesis was accompanied

by a more effective respiratory control with oxidation of substrates for site I, of the ETC (Figure

16

2). In addition of the enhanced substrate utilization, liver mitochondria during FAA present an

elevated oxidative power for reducing equivalents, as it is shown by the higher activity of

NADH-shuttle systems (Figure 3). Both endogenous and reconstituted shuttle systems presented

significant increased activity during the expression of the FEO. The latter is a direct measurement

of the optimal cytosolic NADH oxidative capacity by the liver mitochondria, whereas the

significance of the former must consider alternative routes to transport NADH into the

mitochondria, since hepatic mitochondria is not able to accomplish this process. A possibility in

our experimental protocol is the activation of the fatty acid shuttle, since the expression of the

OSA during the times tested promotes an intense fatty acids mobilization and high synthesis of

ketone bodies (Escobar et al., 1999). The fatty acid shuttle can function in absence of any

cytoplasmic component (Cederbaum et al, (19732).

Mitochondrial swelling is a parameter that depends on ion transport and on the flow of

energy from the ETC. Changes in this activity are suggestive of alterations in membrane

permeability and are predictive of structural modifications in mitochondrial membranes

(Hernández-Muñoz et al., 1992). As to mitochondrial swelling, the only alteration detected in our

experimental protocol was the augmentation in the group of 24 h fasted rats (Figure 5). The

modification involved a difference of 40% and 15% in relation to the ad-libitum and RFS gropus,

which are much lower that the change in mitochondrial swelling induced by chronic CCl4

administration (100%) (Hernández-Muñoz et al., 1992). We do not have an explanation regarding

the modification shown by the 24 h fasted rats. Both parameters, oxidative capacity and

phospholipid and acyl composition in liver mitochondria have been demonstrated to be altered in

fasting conditions (Vendemiale et al., 2001). However, it is interesting to consider that the

increase in mitochondrial swelling was not present in the rats with food restriction at 11:00 h, in

17

particular when this group has a 23 h fasting.

One evidence that RFS produced a new biochemical status in the liver mitochondria, was

the persistent increase in mitochondrial ∆ψ, and hence ∆µ, in the three experimental time points

considered in this study (Table III). Among the parameters that increase the mitochondrial ∆ψ

(Kadenbach, 2003), and compatible with the metabolic adaptations present in the liver during the

expression of the FEO, are: 1) High ATP/ADP ratio; 2) Dephosphorylation of cytochrome c

oxidase at subunit I; 3) presence of 3,5-diiodo-L-thyronine and 4) high levels of palmitate.

Hyperthyroidism induces an enhancement in the energetic status of the mitochondria by

incrementing the ADP/ATP and by stimulating lipid catabolism in the liver (Goglia et al, 1999).

More experiments are needed to define the exact mechanism responsible of the increase in the

liver mitochondrial ∆ψ in our experimental protocol. Taken together, these results are a sign of

an activation in the oxidative and phosphorylative activities of liver mitochondria in rats under

RFS. Remarkably, the activation of the mitochondrial metabolism takes place previous to meal

time, when the FAA is vigorous, and confirms the notion of “biochemical anticipation” of hepatic

physiology before feeding, consisting in a glucogen decrease and an enhancement of lipids

catabolism and mobilization as described by Díaz-Muñoz et al (2000).

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 (Stephan 2002), are highly suggestive of rheostatic

adaptation. Rheostasis, formally homeorhesis, was originally defined as the orchestrated changes

for priorities of a physiological state (Bauman and Currie, 1980). Rheostatic control involves the

coordination of metabolism resulting in the direct portioning of nutrient utilization for

physiological processes such as lactation, pregnancy and growth (Bauman and Currie, 1980).

18

Thus, rheostatic mechanisms provide regulation during changes of state, while 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 two hours, 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 fasting control, since the

RFS rats spend 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 show a metabolic and physiological pattern that is very different from those present in the

ad libitum fed and 24 h fasting groups (Díaz-Muñoz et al., 2000, Aceves et al., 2003, and

Martínez-Merlos et al., 2004). 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 in spite of the 22 h fasting interval (Martínez-Merlos et al., 2004).

The emptying of the stomach approximately 3 h prior to the food access (in our experimental

protocol between 09:00 and 10:00 h), is coincident with a modification in the bioenergetics of the

liver, since it is a change from handling of sugars to lipids as hepatic metabolic fuels (Escobar et

al., 1998). 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

19

activities for lipids (Báez-Ruiz et al., 2005). 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 food entrained oscillator (FEO), independent of the SCN. The anatomical

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 (Stephan 2002).

Independently of the controversy about the precise relevance about the role that the liver

plays in the physiology of the FEO (Davidson et al., 2003, Escobar et al., 2002, Mistlberger

1994, Stephan 2002), 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 increment of ATP, enhancement of acetoacetate indicating an oxidized

redox state, and modulation in the hepatic thyroideal status, point to a direct role played by the

liver mitochondria in the liver physiology of food-restricted rats (Díaz-Muñoz et al., 2000, and

Aceves et al., 2003).

In conclusion, present results provide evidence that liver mitochondrial activity is

modulated by restricted feeding schedules, showing an enhanced oxidative and phosphorylative

capacity specially during the onset of food anticipatory activity. The data also straighten the

anticipatory role of liver as the principal organ in charge of the handling and distribution of

nutrients, in the 2 h interval of food availability. It remains to postulate a model in which all the

biochemical, physiological and endocrinological modifications accounted to RFS induces the

onset of FEO.

20

ACKNOWLEDGMENTS

Authors want to thank to Ms. Fátima Escalera, Maya Rocha and Sabina Wischin for help

and participation in some experiments and statistical analysis, and Dr. Rolando Hernández-

Muñoz (Instituto de Fisiología Celular, UNAM), for technical advice and provision of reactants.

Financial support was obtained from project IN 222102 from DGAPA, PAPIIT, UNAM.

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25

FIGURE LEGENDS

Figure 1

Scheme with the principal mitochondrial metabolic pathways related to ATP synthesis.

See text for explanation.

Figure 2

Mitochondrial Respiratory Control

Mitochondrial respiratory control (state3 / state4) of rats under restricted feeding schedule. Panel

A, respiratory control obtained with glutamate-malate as substrate (10 and 1 mM, respectively)

for site I. Panel B, respiratory control obtained with succinate (5 mM) as substrate for site II. Rats

maintained under a restricted feeding schedule (�), ad-libitum controls (○), and 24 h fasting

(▼). Experimental groups were killed before (08:00 h), during FAA (11:00 h) and 2 h after meal

time (14:00 h). The control group with a 24 h fast was killed at 11:00 h. Results are expressed as

mean + SEM of 6 independent determinations. Significant difference between RFS and ad-

libitum groups (*) and different from 24 h fasting group (x). Tukey´s post hoc test, α = 0.05.

Figure 3

Mitochondrial Shuttles Activities

Mitochondrial NADH-shuttle activities of rats under restricted feeding schedule. Panel A and B,

endogenous and reconstituted aspartate-malate shuttle activity. Panel C and D, endogenous and

reconstituted α-glyserophosphate shuttle activity. White bar, ad-libitum group, black bar, RFS

group, and gray bar 24 h fasting group. Experimental groups were killed before (08:00 h), during

FAA (11:00 h) and 2 h after meal time (14:00 h). Results are expressed as mean + SEM of 6

independent determinations. Significant difference between RFS and ad-libitum groups (*),

26

within the same experimental group (+), and different from 24 h fasting group (x). Tukey´s post

hoc test, α = 0.05.

Figure 4

Mitochondrial Membrane Potential

Mitochondrial ∆µ of rats under restricted feeding schedule. ∆µ was calculated according to ∆µH+

= ∆Ψ + 2.303RT ∆pH /F. Rats maintained under a restricted feeding schedule (�), ad-libitum

controls (○), and 24 h fasting (▼). Experimental groups were killed before (08:00 h), during

FAA (11:00 h) and 2 h after meal time (14:00 h). Results are expressed as mean + SEM of 6

independent determinations. Significant difference between RFS and ad-libitum groups (*) and

different from 24 h fasting group (x). Tukey´s post hoc test, α = 0.05.

Figure 5

Mitochondrial Swelling

Mitochondrial swelling of rats under restricted feeding schedule. Swelling was determined by the

difference between initial and final readings at 546 nm after 5 min of recording. Rats maintained

under a restricted feeding schedule (�), ad-libitum controls (○), and 24 h fasting (▼).

Experimental groups were killed before (08:00 h), during FAA (11:00 h) and 2 h after meal time

(14:00 h). Results are expressed as mean + SEM of 6 independent determinations. Significant

difference from 24 h fasting group (x). Tukey´s post hoc test, α = 0.05.

27

FIGURE 1

28

FIGURE 2 Time (h)

8:00 11:00 14:00

Res

pira

tory

Con

trol

Site

II(s

tate

3/s

tate

4)

3

4

5

6

B8:00 11:00 14:00

Res

pira

tory

Con

trol

Site

I(s

tate

3/s

tate

4)

3

4

5

6

x* A

29

Time (h)

FIGURE 3

0

5

10

15

20

25

30

08:00 11:00 14:00Fast

*+

+*

+

xA

0

30

60

90

120

150

180

08:00 11:00 14:00Fast

B x*+

*+

+

0

5

10

15

20

25

30

08:00 11:00 14:00Fast

*+

Cx*+

*x

0

30

60

90

120

150

180

08:00 11:00 14:00Fast

D

*

+

x*+

+

NA

DH

decr

ease

(nm

oles

/mg

prot

ein

/h)

30

Time (h)

FIGURE 4

Prot

on-m

otiv

efo

rce

(mV)

8:00 11:00 14:00180

200

220

240

260

280

300x** *

31

Time (h)

FIGURE 5

8:00 11:00 14:00

Opt

icde

nsity

diffe

renc

e

0.02

0.03

0.04

0.05

0.06

0.07

x

32

TABLE 1

Parameters of mitochondrial recovery from rat liver

CONDITION

PARAMETER TISSUE FRACTIONS ad lib 8:00

ad lib 11:00

ad lib 14:00

Fasting (24 h)

FRS 8:00

FRS 11:00

FRS 14:00

Homogenate U/mg/min 13 + 1.0 14 + 1.0 13 + 1.2 12 + 1.3 15 + 1.4 15 + 1.6 14 + 1.7

Citrate synthase activity Mitochondria

U/mg/min 71 + 10.4 72 + 2.9 70 + 8.5 75 + 2.0 76 + 3.7 78 + 3.8 77 + 1.9

Homogenate mg/g of wet tissue 206 + 11 205 + 21 200 + 21 210 + 20 221 + 18 238 + 25 236 + 26

Mitochondrial (yield) mg/g of wet tissue 6.8 + 0.4 7.6 + 0.5 6.9 + 0.1 7.0 + 0.5 7.8 + 0.4* 8.7 + 0.5* + 7.7 + 0.5*

Mitochondrial (calculated total)

mg/g of wet tissue 37.7 + 3.9 39.9 + 2.4 37.3 + 5.9 33.6 + 3.8 43.6 + 4.1 46 + 4.2 43 + 4.7

Protein content

Recovery (%) 18 + 1.7 19 + 1.3 18 + 2.8 18 + 1.9 18 + 2.0 19 + 2.2 18 + 2.1

Results are expressed as mean + SEM of 6 independent determinations. Enzyme activities were calculated as described in methodology. Total calculated mitochondrial protein and mitochondrial yield were calculated according to Idell-Wenger, et al (1982). *P< 0.05 versus ad lib controls; +P< 0.05 against fasted group.

33

TABLE 2

Parameters of mitochondrial oxidation of substrates for sites I and II in rats under food-restricted schedules.

SITE I

DNP (natoms

O2/min/mg)

ADP/O

ATP Synthesis(nmol/min/mg)

Fast (24h) 108 + 2.5 2.4 + 0.1 140 + 22

08:00 95.3 + 3.5 2.5 + 0.1 136 + 7.8 11:00 95.0 + 3.6 2.6 + 0.1 128 + 8.8 ad lib14:00 96.4 + 4.3 2.5 + 0.1 129 + 4.2

08:00 108 + 2.5 2.6 + 0.1 147 + 3.4

11:00 122 + 3.6* 2.7 + 0.1 214 + 20* +RFS

14:00 103 + 1.5 2.6 + 0.1 151 + 13

SITE II DNP

(natoms O2/min/mg)

ADP/O ATP Synthesis(nmol/min/mg)

Fast (24h) 100 + 2.6 1.5 + 0.2 104 + 1.9

08:00 124 + 2.6 1.7 + 0.1 126 + 3.2 11:00 123 + 2.5 1.6 + 0.1 121 + 4.6 ad lib14:00 127 + 4.3 1.6 + 0.1 120 + 6.2

08:00 148 + 3 1.8 + 0.1 159 + 13

11:00 156 + 15* + 1.8 + 0.2 197 + 14* +RFS

14:00 134 + 2.4 1.7 + 0.1 130 + 10

Experimental groups were killed before (08:00 h), during FAA (11:00 h) and 2 h after meal (14:00 h). Results are expressed as mean + SEM of 6 independent determinations. Uncoupled state obtained with dinitrophenol (+DNP). ADP/O ratio and ATP synthesis calculations were done as described in Experimental Design section. *P < 0.05 against control group; +P< 0.05 against fasted group.

34

TABLE 3

Modifications of the membrane electrical potential (∆Ψ) and ∆pH from liver mitochondria during FEO expression

∆Ψ ∆pH

Fasted 24 h 194.8 + 1.8 102 + 1.8

08:00 191.2 + 1.2 99 + 5.6

11:00 204.2 + 1.7 102 + 2.6 ad lib

14:00 205.8 + 1.5 105 + 3.2

08:00 281.2 + 3.0* 103 + 2.3

11:00 284 + 1.2* + 117 + 1.8* +RFS 14:00 278.2 + 1.3* 110 + 2.3

NOTE: ∆ψ and ∆pH were calculated by radiometric assays (See Experimental Design section). The ∆Ψ was calculated from the distribution of (TPP+2) as the ∆pH from [14C]acetate distribution, according to the Nernst equation (see Calculations and statistics). *P < 0.05 against control group; +P< 0.05 against fasted group.

35

TABLE 4

F values obtained with the two-way ANOVA for independent measures, for main metabolic parameters for RFS group and its ad libitum controls.

Feeding Condition Time Interaction

F(1,16) P F(3,16) P F(3,16) PMitochondrial protein 63.7 <0.001 4.1 NS 0.63 NS

R..C. Site I 20.3 <0.001 4 NS 6.2 NS

R C. Site II 33.5 <0.001 29.3 NS 14.2 <0.001 Swelling 8.1 NS 0.85 <0.05 0.43 NS

Mal-asp. Shuttle* 34.8 <0.001 9.4 <0.05 8.6 <0.05

α-gly. Shuttle* 233 <0.001 28 <0.001 18.8 <0.001

∆µH+ 10 <0.001 7.3 <0.001 9318 <0.001

R.C. = respiratory control; * = reconstituted activity, and NS = not significant

36

TABLE 5

F values obtained with the one-way ANOVA for comparison between the 24 h fasted and the RFS-11:00 h groups

F(2,15) P Mitochondrial protein 14.15 <0.05

R.. C. Site I 17.08 NS R.. C. Site II 40 <0.001

Swelling 6.2 NS

Mal-asp. shuttle* 15.5 <0.001

α-gly. shuttle* 62 <0.001

∆µH+ 941 <0.001

R.C. = respiratory control; * = reconstituted activity, and NS = not significant