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Lack of Effect of Caloric Restriction on Bioenergetics and Reactive Oxygen Species Production in Intact Rat Hepatocytes

School of Biological Sciences, University of Liverpool, United Kingdom.

Address correspondence to Adrian J. Lambert, Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY, UK. E-mail: adrian.lambert{at}mrc-dunn.cam.ac.uk

	Abstract

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To investigate the hypothesis that caloric restriction alters mitochondrial function in situ, intact hepatocytes were isolated from fully fed and calorie-restricted (55% of control food intake, 4 months duration) male Brown-Norway rats at 6 months of age, and various parameters were determined. Overall, the production of reactive oxygen species was not affected by caloric restriction, neither were the mitochondrial membrane potential, oxygen consumption driving proton leak, or oxygen consumption driving ATP turnover. It is concluded that while isolated mitochondria from liver tissue of calorie-restricted animals display a reduction in the generation of reactive oxygen species, it was not possible to confirm this effect in isolated hepatocytes. Further work is required to establish what effect, if any, caloric restriction has on the rate of generation of reactive oxygen species in intact cells and tissues and importantly at the whole-animal level.

A plausible mechanism by which caloric restriction could lower ROS production by mitochondria in vitro was recently proposed (6). The mitochondrial protonmotive force (p) is the electrochemical potential produced by proton pumping by the electron transport chain, it is consumed by ATP synthase to produce ATP and by proton leak to produce heat. Changes in the rate of either ATP synthesis or proton leak will affect p and the rate of oxygen consumption. It is known that the ROS production rate by mitochondria is sensitive to the magnitude of the protonmotive force (p) (14–16). It was demonstrated that short-term caloric restriction (4 months duration) resulted in a significantly lower p, which would explain, at least in part, why caloric restriction resulted in significantly lower ROS generation. The important question that arises from this work is, are the same or similar mechanisms operating in mitochondria in vivo? Clearly, if the effects of caloric restriction such as lowered ROS production in isolated mitochondria are not induced in the living animal, then lowered ROS production cannot be involved in the anti-aging mechanism of caloric restriction. At present, there is very little information concerning the effects of caloric restriction on mitochondrial function in intact cells and none on the in vivo situation for mammals. We report here the results of a short-term study designed to address this issue in isolated cells derived from CR rats.

	METHODS

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Animals, Feeding, and Isolation of Hepatocytes
Male Brown-Norway rats were used in this study. All animal husbandry and procedures involved were in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. CR rats were housed singly, and the intake of the pelleted diet was limited such that body weights were maintained at 55% of the age-matched fully fed rats. Caloric restriction was initiated at 60 days of age. The restricted diet was preweighed and supplied between 10:30 and 11:00 AM. CR rats consumed most of their food within about 5 hours, effectively making them daytime meal-eaters. However, ad libitum-fed rats tend to feed during the dark cycle. Therefore, the fully fed control rats in this study (also housed singly) were given free access to food between 10:00 AM and 3:00 PM to synchronize them to the same feed/fast cycle as the CR rats. The ages of the animals used in this study were 6.2 ± 0.1 months (means ± standard errors, n = 4) and thus the duration of caloric restriction was 4 months.

Cells were prepared by a modification of the two-step collagenase perfusion technique as described by Seglen (17). All isolations were performed between 9 and 9:30 AM, before the animals were fed. The viability of isolated cells was assessed by the exclusion of trypan blue. Small aliquots of cells were dried to constant mass at 60°C to determine their dry weight.

Standard Incubation Protocol
Hepatocytes were incubated at 5–10 mg dry weight per milliliter with shaking (100 cycles/min) at 37°C in a humidified atmosphere of 95% air/5% CO₂. The buffer was Hank's balanced salt solution (HBSS) containing 10 mM HEPES, 0.05% (w/v) bovine serum albumin (BSA), 10 mM NaHCO₃, 10 mM lactate, and 1 mM pyruvate (pH 7.4). Triphenylmethyl phosphonium (TPMP; 1 mM) and 0.1 mg/ml inulin were also present as carriers for [¹⁴C]TPMP and [¹⁴C]methoxyinulin, respectively. Cells were preincubated for 10 minutes to allow all ion gradients to reach equilibrium. Various probes were then added to measure ROS production (dichlorodihydrofluorescein diacetate[(H₂DCFDA]) or membrane potentials (³H₂O, [¹⁴C]methoxyinulin, ³⁶Cl^–, [¹⁴C]TPMP, ⁸⁶Rb⁺), and the incubations were continued for a further 20 minutes. Inhibitors and/or uncouplers (oligomycin, valinomycin, myxothiazol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone [FCCP]) were also added to specific incubations as described below. All incubations were performed in triplicate.

Measurement of Oxygen Consumption
Following the standard incubation period, aliquots of cells were taken and oxygen consumption was measured using a Clark-type oxygen electrode (Rank Brothers, Cambridge, U.K.). Oxygen consumption rates were determined in the presence and absence of oligomycin, to allow calculation of total and proton leak-dependent oxygen consumption. Additional runs were performed with 100 µM valinomycin, 3 µM myxothiazol, 10 mg/ml oligomycin, and 20 µM FCCP. These conditions allowed the nonmitochondrial oxygen consumption to be determined, as mitochondria are unable to oxidize substrates and synthesize ATP, and are completely uncoupled.

Measurement of ROS Production
Cells were incubated as described above with 10 mM H₂DCFDA as a probe for ROS production (18). Following the incubation period, aliquots of cells were quickly transferred to an ice-cold microfuge tube and centrifuged (1000 x g at 2°C) for 30 seconds. An aliquot of the supernatant was then quickly transferred to a cuvette and allowed to reach room temperature; the fluorescence was determined at excitation and emission wavelengths of 488 and 525 nm, respectively. Nonspecific background signals were obtained by incubation with cells but without probe, then by incubation with probe but without cells, and subtracted accordingly. Measurements were performed in the absence or presence of oligomycin. Additional runs were performed with 100 µM valinomycin, 3 µM myxothiazol, 10 mg/ml oligomycin, and 20 µM FCCP to determine mitochondrial independent ROS production.

Measurement of Mitochondrial Membrane Potential, _m
Mitochondrial membrane potential in intact hepatocytes was determined as described (19,20) using equation 1:

where V_c and V_m are the proportions of the cell volume occupied by cytoplasm and by mitochondria, respectively, and were estimated by electron microscopy. a_m, a_c, and a_e are the TPMP binding correction factors in the mitochondrial matrix, cytoplasm, and external medium, respectively. a_m was determined using isolated mitochondria (6,19), and a_c was measured by comparing the accumulation ratios of [¹⁴C]TPMP and ⁸⁶Rb⁺ (21). a_e was assumed to be 1 because the BSA concentration in the medium was low. The subscripts "in" and "out" refer to the concentrations of isotopic probe in the cells and in the medium, respectively. [Cl^–]_in/[Cl^–]_out and plasma membrane potential (_p) were determined from the distribution of ³⁶Cl^–. [TPMP]_in/[TPMP]_out was determined from the distribution of [¹⁴C]TPMP. Hepatocyte volume was determined using ³H₂O as a cell-permeant probe and [¹⁴C]methoxyinulin as an impermeant probe.

At the end of the incubation period, aliquots (0.7 ml) of cells were taken and added to 1.5 ml microfuge tubes containing 350 µl of silicone oil (42% (v/v) dinonylphthalate and 58% DC 550 silicone fluid) layered over 100 µl of 2% (v/v) TX-100 in 250 mM sucrose. The tubes were then centrifuged at 11,000 x g for 2 minutes at room temperature, and 200 µl of the supernatant was pipetted into a scintillation vial and mixed with 3.5 ml of scintillant. The residual supernatant and most of the oil layer were removed, and the tube walls were wiped dry with twisted tissue paper. The pellet was resuspended, and the bottom of the tube was cut directly into a scintillation tube containing 200 µl of distilled water. Scintillant (3.5 ml) was added, and the tube contents were mixed. The radioactivities of the supernatant and pellet were determined by dual-channel scintillation counting for ³H/¹⁴C for hepatocyte volume and ³H/³⁶Cl for _p with use of appropriate crossover and quench corrections. Radioactivities for ¹⁴C for [TPMP]_in/[TPMP]_out were measured by single-channel counting.

Morphometric Analysis
Aliquots of cells were pelleted and fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate. They were then stained with osmium tetroxide, dehydrated, and embedded in araldite. Electron micrographs of ultrathin sections were prepared using a Philips CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands), and mitochondrial volume (V_m) was determined using a 1-cm grid overlaying the micrographs (20). V_m was calculated as the number of intersections in mitochondria divided by the total number of intersections in cells, and V_c was calculated as the number of intersections in cytoplasm divided by the total number of intersections in cells. The number of intersections on lipid droplets (that do not take up TPMP) was corrected for by subtraction. V_m is the total mitochondrial volume (matrix, cristae, and intermembrane space), but TPMP is only taken up into the matrix; therefore, mitochondrial matrix volume was calculated as 0.565 of V_m (20,22).

Materials
Amersham Pharmacia Biotech supplied ³H₂O, Na³⁶Cl, and ⁸⁶RbCl; [¹⁴C]TPMP was purchased from Moravek Biochemicals (Brea, CA). Dow Corning silicone fluid 550 was purchased from BDH (Poole, Dorset, U.K.). All other chemicals were purchased from Sigma (Poole, Dorset, U.K.).

Statistical Analysis
Results are presented as means ± the standard error of the mean. The significance of differences between group means was assessed using Student's t test. Values of p <.05 were considered to be significant.

	RESULTS

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It was shown previously that short-term caloric restriction (55% reduction in food intake for 4 months) in young adult (6 months of age) male Brown-Norway rats resulted in a significant reduction in both liver mitochondrial ROS production and protonmotive force (6), but state IV oxygen consumption rate remained unchanged. To determine if these effects of caloric restriction were also manifest in situ, ROS production and protonmotive force were measured in intact hepatocytes. As found previously for male Sprague-Dawley rats (23), cell viability was unaffected by caloric restriction (not shown).

Bioenergetic data of hepatocytes isolated from fully fed or CR rats is shown in Figure 1. There were no significant effects of caloric restriction on oxygen consumption (expressed as nmol O₂/min/mg dry weight) or mitochondrial membrane potential (_m) under any of the conditions examined. Thus nonmitochondrial oxygen consumption as measured in the presence of oligomycin, valinomycin, myxothiazol, and FCCP was unaffected by caloric restriction (diamonds in Figure 1). Oxygen consumption driving proton leak at a _m of about 130 mV as measured in the presence of oligomycin was also unaffected by caloric restriction (triangles). Oxygen consumption driving ATP turnover and proton leak (as measured in the absence of oligomycin) at about 125 mV was also not effected by caloric restriction (squares). Subtraction of the nonmitochondrial oxygen consumption rates from the rates driving proton leak and ATP turnover and proton leak did not reveal any effects of caloric restriction. When O₂ consumption rates were normalized to 1 x 10⁶ cells, again there were no effects of caloric restriction under any of the conditions examined (not shown).

View larger version (13K): [in this window] [in a new window]   Figure 1. Bioenergetic data of hepatocytes from fully fed and calorie-restricted (CR) rats. Closed symbols represent fully fed animals; open symbols represent CR animals. Diamonds represent nonmitochondrial O2 consumption and m obtained in the presence of oligomycin, valinomycin, myxothiazol, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP); triangles represent proton leak-driven O2 consumption and m obtained in the presence of oligomycin; squares represent ATP turnover and leak-driven O2 consumption obtained with no additions. See "Methods" for further details. Each point is the mean ± SEM, n = 4 separate animals examined. Animals were 6 months of age, and caloric restriction was initiated at 2 months

View larger version (14K): [in this window] [in a new window]   Figure 2. Reactive oxygen species production in hepatocytes from fully fed and calorie-restricted (CR) rats. Closed bars represent fully fed animals; open bars represent CR animals. Hepatocytes were incubated as described in "Methods" with dichlorodihydrofluorescein diacetate (H2DCFDA) as a probe for reactive oxygen species production. Each bar is the mean ± SEM, n = 4 separate animals examined. Animals were 6 months of age, and caloric restriction was initiated at 2 months. NM = nonmitochondrial (in the presence of oligomycin, valinomycin, myxothiazol, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone [FCCP])

View this table: [in this window] [in a new window]   Table 1. Parameters Related to m in Hepatocytes From Fully Fed Rats and Calorie-Restricted (CR) Rats.

	DISCUSSION

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It is possible that production of ROS by mitochondria is a major factor in the aging process, ROS production rate by isolated mitochondria is lowered by caloric restriction (4–9,26,33), and that there is a strong correlation between maximum life span of different species and mitochondrial ROS production rate (34,35). There are several factors that may explain why mitochondria from CR animals produce ROS at lower rates, one of these is the magnitude of the mitochondrial membrane potential, p. Consistent with the "uncoupled and surviving" theory of aging (36,37), it was demonstrated that caloric restriction lowers p in isolated liver mitochondria (6); this may either explain totally or at least in part why caloric restriction lowers ROS production. The objective of this study was to ascertain if a similar adaptation could be demonstrated in mitochondria in isolated hepatocytes.

The values of the parameters required to calculate _m (in Table 1) are in good agreement with the values reported in the literature. Thus the reported values for cell volume are 1.2–1.7 µl/mg dry weight (21,38); reported values for _p are around –30 mV (21,38–41); reported values for V_m and V_c are approximately 0.2 and 0.8, respectively (21,22,41), and a_c has published values of about 0.2 (21,41).

The reported values of _m in rat hepatocytes are approximately –150 mV (21,30,38,40). The values presented here are slightly lower than this, but given the good agreement of the other parameters (Table 1) and oxygen consumption rates ± oligomycin with those in the literature, this is probably a system effect as opposed to a biological effect, and would apply to cells from both fully fed and CR animals equally. It could not be demonstrated in hepatocytes isolated from short-term CR-fed animals that mitochondrial membrane potential is significantly lower than that seen in fully fed controls. This is in contrast to the finding that, in isolated liver mitochondria from CR animals, overall protonmotive force (p) is significantly reduced (6). It may be that the chemical (pH) component of p (p = _m + pH) is lower in intact cells from CR rats, and in situ p is lower, but as yet there is no evidence that this is so. This preliminary finding that _m is unchanged by caloric restriction in whole cells is consistent with the finding that the production of ROS in whole cells was also not effected by caloric restriction. The production of ROS is highly sensitive to the magnitude of _m (14–16) and pH (16), hence if _m in situ is unchanged by caloric restriction then perhaps it is not surprising that ROS production in situ is unchanged also.

There could be lowered mitochondrial ROS production in cells from CR animals, but the effect is masked by changes in activities of ROS scavenging enzymes. In general, caloric restriction does indeed prevent or ameliorate the age-related changes in enzymes such as Mn- and Cu/Zn-SOD, catalase, and glutathione peroxidase, hence comparison of mature or old animals reveals a significant effect of caloric restriction. However, there do not appear to be any effects of short-term caloric restriction on these enzyme activities in liver (42–44). It seems unlikely, therefore, that differences in antioxidant enzyme activities explain the lack of effect of caloric restriction on ROS generation by intact hepatocytes.

It was found previously that the total ROS production rate (in the absence of electron transport chain inhibitors) by isolated liver mitochondria from young CR rats was about 40% lower than the rate in mitochondria from fully fed controls (7). It was also observed that in the liver of rats on caloric restriction, there is an approximately 40% greater density of mitochondrial protein per gram of liver tissue (45). It may be that these two factors cancel each other, so that per milligram of cells, no effect of caloric restriction on ROS production is seen. Total mitochondrial volume (V_m) was unchanged with caloric restriction (Table 1), so an increase in mitochondrial protein density may be due to an increase in inner mitochondrial membrane surface area.

The rates of oxygen consumption reported in this study are also in good agreement with reported values of 6–8 nmol of O₂/min/mg dry weight (38,41). It appears, therefore, that caloric restriction does indeed have no effect on mitochondrial oxygen consumption driving proton leak and ATP turnover when measured in situ in intact hepatocytes. Hence the finding that caloric restriction does not effect oxygen consumption rate in isolated liver mitochondria (4,26,45,46) also applies to mitochondria in intact hepatocytes.

From the data presented here, it may seem that short-term caloric restriction does not affect liver tissue at all. This is not the case. In mice, short-term caloric restriction does induce changes in liver tissue at the level of transcription (47,48). However, these changes do not appear to manifest themselves in the whole cell as changes in mitochondrial bioenergetics and ROS production. The possibility remains that, in vivo, both ROS generation rate and _m are lower in CR animals, but the effect is lost on isolation of the hepatocytes and incubation under the conditions used. Alternatively, it may be that for liver, the anti-aging mechanisms of caloric restriction are exerted primarily via other pathways, such as enhanced removal or repair of oxidative damage. However, not all evidence supports the oxidative damage theory of aging—despite lower ROS production, we found no effects of caloric restriction on oxidative damage to mitochondrial protein (49). An increase in levels of 8-oxo-2-deoxyguanosine and tumor incidence is reported in mice heterozygous for the SOD2 (manganese superoxide dismutase) gene (50), yet this lifelong reduction in SOD2 activity does not appear to affect aging.

Overall, the results of this study do not support the idea that ROS production is lowered by caloric restriction in intact hepatocytes. Further work is required to confirm that caloric restriction lowers mitochondrial ROS production in situ in other tissues and at the whole-animal level and if so, by what mechanism.

	Acknowledgments

 
This work was funded by the Biotechnology and Biological Sciences Research Council, grant 26/SAG09938.

	Footnotes

 
Decision Editor: James R. Smith, PhD

Received June 2, 2004

Accepted September 7, 2004

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