The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 μM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 μM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.
- firefly luciferase
- bioluminescence assay
- ATP production
- caloric restriction
mitochondria produce ∼90% of the required energy necessary for cellular function (46). The inner membrane of the mitochondria contains a complex series of enzymes and other proteins that are involved in the catabolism of food molecules to liberate metabolically useful energy in the form of ATP. Cellular function is maintained through the hydrolysis of ATP to ADP. The catalytic sites on the F1 head of the ATP synthase complex, which projects onto the protochemically negative matrix side (N-phase) of the membrane, contain three nucleotide binding sites that undergo a multiphase, catalytic cycle to produce ATP (43) from ADP and phosphate (i.e., oxidative phosphorylation). Because ATP is tightly bound to the F1 head in the mitochondrial membrane, a large amount of free energy is released during the rapid hydrolysis of ATP to ADP or AMP. The large free energy released during the hydrolysis of ATP is available for immediate use (i.e., muscle contraction, chemiosmotic homeostasis, and normal cellular function) or can be stored for future cellular activity, such as protein synthesis, genomic replication, and cell growth (37, 43).
To study ATP content and production from isolated mitochondria, we used a commercially available ATP determination kit. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. Upon addition, ATP combines with luciferin to form luciferyl adenylate and inorganic pyrophosphate (PPi) on the surface of the luciferase enzyme as shown in reaction 1 reaction 1
While bound to the enzyme, luciferyl adenylate combines with O2 to form oxyluciferin and AMP (5) through a series of enzymatic redox reactions. As oxyluciferin and AMP are released from the enzyme's surface, a quantum yield of light is emitted in proportion to the ATP concentration as shown in reaction 2 reaction 2
The light emission (hν) can then be recorded and quantified using a chemiluminometer.
This study introduces a quick and reproducible methodology for measuring ATP in isolated mitochondria. The effects of age and caloric restriction on the amount of ATP present as well as the rate of ATP production on isolated mitochondria in the brain and liver have yet to be studied. Hence, we determined in vivo whether ATP content and production change with age and lifelong caloric restriction in the brain and liver. During aging, the production of reactive oxygen species in the mitochondria may lead to a reduction in the activity of the respiratory chain complexes, and hence a decline in ATP production (2, 20, 26, 47). In contrast, caloric restriction may be able to reduce the age-related increase in oxidative damage to the respiratory complexes.
Chemicals. The ATP determination kit (A-22066) by Molecular Probes (Eugene, OR) containes d-luciferin, luciferase [40 μl of a 5 mg/ml solution in 25 mM Tris·acetate, pH 7.8, 0.2 M ammonium sulfate, 15% (vol/vol) glycerol, and 30% (vol/vol) ethylene glycol], dithiothreitol (DTT), ATP, and a reaction buffer (10 ml of 500 mM tricine buffer, pH 7.8, 100 mM MgSO4, 2 mM EDTA, and 2 mM sodium azide). The reagents and reaction mixture were combined according to the protocol by Molecular Probes; however, the volume of each assay was increased to 1,000 μl to account for the ∼100-fold stoichiometric increase in the quantum yield due to the higher optical density (OD) readings for the production of ATP relative to the content. In addition, 1 mM pyruvate and 1 mM malate were added to the reaction mixture as substrates for oxidative phosphorylation (14, 48).
Animals. Young ad libitum-fed (young, n = 8, 12-13 mo), old ad libitum-fed (old, n = 6, 26-28 mo), and old caloric-restricted (CR, n = 6, 27-28 mo) male Fischer 344 rats [National Institute of Aging (NIA) colony, Harlan Sprague-Dawley, Indianapolis, IN] were used. Caloric restriction was started at 3.5 mo of age (10% restriction), increased to 25% restriction at 3.75 mo, and maintained at 40% restriction from 4 mo throughout the individual animal's life. The rats were housed one per cage in a temperature-controlled (18-22°C) and light-controlled environment with a 12:12-h light-dark cycle. Following 1 wk of acclimation after arrival from the NIA, one animal was randomly killed daily, after being anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). All treatment of animals throughout this study conformed fully to the “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society. In addition, protocols received local institutional animal care committee approval.
Mitochondrial isolation and protein concentration. Intact mitochondria from the liver and frontal brain cortexes were immediately isolated using differential centrifugation. The tissue from the liver was homogenized (on ice) in 1:10 wt/vol of ice-cold buffer A [0.220 M mannitol, 0.070 M sucrose, 0.5 mM EGTA, 2 mM HEPES (pH 7.4) and 0.1% fatty acid free BSA] using a Potter-Elvehjem glass-glass homogenizer. The homogenate from the liver was centrifuged (Eppendorf 5810R, Brinkmann Instruments) at 1,600 g (4°C) for 10 min, and the resulting supernatant was centrifuged at 18,000 g (4°C) for 10 min. The pellet was resuspended in buffer A and centrifuged at 18,000 g (4°C) for an additional 10 min. The tissue from the frontal cortex of the brain was homogenized as before but in ice-cold suspension (1:10 wt/vol) buffer B [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.1 mM PMSF] (38). The homogenate from the frontal cortex was centrifuged at 750 g at 4°C for 5 min. The supernatant was then centrifuged at 8,000 g for 20 min at 4°C. The pellets from the liver and frontal cortex were resuspended in their respective buffers, and the freshly isolated mitochondria were used immediately for the analysis of ATP content and rate of ATP production. Mitochondrial protein concentration was determined using the Bradford method (4).
Standard curve for ATP determination. For all experiments, ATP standard curves were run in the range of 0.5 to 50 μM (0.5, 1.0, 5.0, 10.0, 25.0, and 50.0 μM, respectively). The standard curve was linear up to a concentration of 25.0 μM; however, the curve started to deviate from linearity as the concentration approached 50.0 μM. Hence a second-order polynomial trend line was used to fit the data due to apparent deviations from the Beer-Lambert law. In all experiments, the correlation coefficient was 0.990 or higher.
Stability of ATP. The stability of the reaction mixture as well as two relevant ATP standards, which gave luminescent (OD) readings comparable in magnitude to median luminescent readings for ATP content, 0.5 μM, and rate of production, 25.0 μMina10 μl mitochondria sample, were measured (see ATP content and production methodology). Measurements were recorded every 5 s for a total analysis of 5 min.
Inhibition of ATP production. To test the functionality of the ATP determination assay, 0, 1, and 5 mM oligomycin were added and incubated for 10 min at 37°C to freshly isolated mitochondria. The inhibition in the rate of ATP production was determined by comparing the oligomycin-treated mitochondria to an equal portion of freshly isolated mitochondria from the same tissue.
ATP content and production methodology. All solutions were placed in a heating block and maintained at a temperature of 28°C that was, kinetically, the optimum temperature (Molecular Probes) for the reaction mixture. Analyses began on the Turner Designs TD 20/20 (Sunnyvale, CA) luminometer using a blank (deionized H2O) to correct for instrumental drift and possible physical interferences within the polypropylene cuvettes (12 × 50 mm, Turner Designs, Sunnyvale, CA). Next, the luminescent signals for six ATP standards ranging from 0.5 to 50 μM (0.5, 1.0, 5.0, 10.0, 25.0, and 50.0 μM, respectively) were measured. Initially, for each standard, background measurements of the reaction mixture (990 μl) were recorded. Then, 10 μl of each standard was added to each of the reaction mixtures, and the luminescence was recorded. After the standards were completed, the luminescence of 990 μl of the reaction mixture was recorded. To this mixture, 10 μl of isolation buffer and 10 μl of 2.5 mM ADP were added and recorded so that this background associated with the ADP solution could be subtracted from the measurements of the mitochondria used to determine the maximum ATP production. Measurements were obtained every 5 s for a total analysis time of 30 s (a total of 6 measurements) for each mixture or subsequent addition. Because all of these measurements were stable over the entire analysis, the six ADP background measurements were averaged, and the mean was used in the calculations. Because the sensitivity of the standards and reaction mixture decreases over prolonged time, all of the standards were analyzed during isolation of the mitochondria to maximize the luminescent signal.
The measurements for ATP content and rate of production were made immediately after isolation of the mitochondria to improve the accuracy of the measurements and reduce any inherent errors associated with the luminescent decay or reduced viability of the isolated mitochondria. The mitochondria were kept on ice to ensure this added stability and were not incubated at 28°C (consistent with the standards) or biological temperature (∼37°C); however, this should have no effect on the accuracy because thermodynamic laws of heat transfer dictate that the small volume (10 μl) of cold mitochondria will have virtually no effect on the overall temperature of the larger assay volume of the reaction mixture (990 μl), which is kept at 28°C. Like the standards, the background of the reaction mixtures (990 μl) was recorded before the addition of mitochondria. Afterward, 10 μl of freshly isolated mitochondria from the frontal cortex of the brain or liver were added to the reaction mixture to determine the ATP content. The analyses were carried out and recorded similarly to the aforementioned standards. The luminescent signal decreased rapidly during each successive reading as the ATP was quickly converted through a series of enzymatic redox reactions to AMP through the release of the two terminal phosphate groups (see reactions 1 and 2). Thus only the first reading was used to calculate the ATP content in the mitochondria because it corresponded to the maximum amount of ATP present [and hence yielded the highest OD reading on the luminometer of the 6 measurements]. Immediately after the ATP content measurements, 10 μl of 2.5 mM ADP was added to the cuvette containing the reaction mixture and mitochondria to determine the rate of ATP production. ADP (2.5 mM) was used because it corresponds to a physiological concentration found in myocytes (33). The amount of ATP produced within the mitochondria is directly proportional to the ADP available, so any change in ADP concentration should directly affect the ATP concentration. Hence, it is supportive that ATP content and rate of production varied in unison, because production and consumption are regulated by ATP-to-ADP ratio (11). The system was monitored as previously mentioned, and the final reading was used to determine the rate. The values for ATP content and rate of production were normalized to the mitochondrial protein concentration present in each 10-μl aliquot. All mitochondrial samples were performed in triplicate, and an average of these results was used in the quantification of the ATP content and rate of production.
Statistical analysis. Unpaired t-tests were used for comparisons between groups, Pearson product-moment correlations were performed between dependent variables, and the intra- and interassay coefficients of variation were calculated using a statistical package from Prism (San Diego, CA). A P value of <0.05 was considered significant.
Reaction mixture and standards stability. Wibom et al. (48) studied the stability of mitochondrial preparations in pyruvate and malate and found a linear decrease in activity (i.e., mitochondrial degradation) of 11 ± 2%/h over a 5-h period. While the activities of the mitochondrial preparations were not measured, the overall stability of the mitochondria to produce ATP was apparent in this study as the readings tended to decrease for subsequent measurements. As a result, we measured the decline in the sensitivity of the reaction mixture as well as two relevant standards with time. For the reaction mixture (Fig. 1A), the linear decay was 4.88 × 10-3 U/min or a 2.16% decay/min. Because the OD values for the reaction mixture are much smaller than the ATP OD readings, the inherent decay is negligible. For a 0.5 μM ATP standard (Fig. 1B), i.e., OD readings consistent in magnitude with the luminescent readings for the ATP content present in 10 μl of mitochondrial sample before normalization to the protein concentration, the luminescent signal underwent a linear decay (r2 = 0.988) for the entire 5 min of analysis. The signal decayed at a rate of 5.70 U/min or 1.39% decay/min. Because the amount of ATP present in the tissue of interest is analyzed over a 30-s interval, this small linear decay can also be considered negligible. For a 25-μM ATP standard (Fig. 1C), OD readings consistent with the luminescent readings for rate of ATP production in 10 μl mitochondrial sample before normalization to the protein concentration, the luminescent signal underwent a logarithmic decay defined by Eq. 1 1 where r2 = 0.993.
Hence during a 30-s analysis on the rate of ATP production, the luminescent signal corresponding to this high concentration would decay by 13.4%.
Inhibition of the F0F1-ATPase enzyme by oligomycin. To confirm the functionality of the ATP assay, 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation, were incubated with a portion of the isolated mitochondria for 10 min at 37°C and compared with a control (equal portion of freshly isolated mitochondria from the same sample). The 1 and 5 mM oligomycin were able to significantly inhibit the production of ATP by 34 and 83% (1 mM, P = 0.0001; 5 mM, P < 0.0001), respectively (Fig. 2). All tissues were run in triplicate (n = 4), and the average of these results was used to determine the extent of inhibition to the production of ATP.
Effects of age and caloric restriction on body and tissue mass and ATP content and rate of production in liver and brain. As expected, body mass decreased by 15% with age (young vs. old, P = 0.002) and an additional 33% through lifelong caloric restriction (old vs. calorie restricted, P < 0.0001) (Table 1). In the brain and liver, there was no significant change in mass due to age. In striking contrast, caloric restriction led to a 41% (old vs. calorie restricted, P < 0.0001) reduction in the liver mass while having no effect on brain weight (Table 1).
Previously, we showed that with age, ATP content and production decreased by ∼50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria (11). However to date, no one has quantitatively measured in vivo the effects of age and caloric restriction on the ATP content or on the rate of ATP production in the isolated mitochondria of the brain or liver of 12- to 13-mo-old ad libitum-fed, 26- to 28-mo-old ad libitumfed, and 27- to 28-mo-old lifelong caloric-restricted rats. Consequently, measurements were made on two separate tissues: brain and liver (Table 2). In the brain, the ATP content was unaffected by age or caloric restriction (Fig. 3A). Moreover, the rate of ATP production was not different due to age and caloric restriction (Fig. 3B). In the liver, the results showed a similar trend: neither age nor caloric restriction had any significant effect on the ATP content (Fig. 3C) or on the rate of ATP production (Fig. 3D). However, the lifelong calorie-restricted rats tended to have a higher production rate of ATP in the liver, but these changes where not statistically significant (Table 2).
Recently, we determined the rate of ATP production in skeletal muscle and the heart (complete data published in Ref. 11). The rate of skeletal muscle ATP production was 7.29 ± 1.01 nmol ATP·mg protein-1·min-1 in 12-mo-old ad libitum-fed animals compared with 3.17 ± 0.99 nmol ATP·mg protein-1·min-1 in the 26-mo-old animals. In the heart mitochondria there were no significant changes with age: 18.31 ± 1.46 (12 mo old) vs. 17.92 ± 2.02 nmol ATP·mg protein-1·min-1 in 26-mo-old rats. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. The production of ATP in skeletal and heart muscle mitochondria was 2.77 ± 0.53 and 18.86 ± 1.84 nmol ATP·mg protein-1·min-1 for 26-mo-old caloric-restricted rats (11). When the rate of ATP production of the brain and the liver (Table 2) are compared with that of the heart and skeletal muscle (gastrocnemius), the data show that the heart has the greatest rate of production: heart > liver > brain > gastrocnemius muscle.
Intra- and interassay coefficients of variation to test precision and variability. To test the precision and variability of the aforementioned methodology to measure ATP content and production in isolated mitochondria, we determined the intra- and interassay variation coefficients. The intra-assay variation coefficient, a measure of the precision of the assay, was determined by measuring the average of the triplicate readings for each sample (Table 3). The SD of each triplicate reading was divided by its average and multiplied by 100 to create a percentage (%CV). The calculated %CV was then averaged for each animal (n = 20 total animals) to determine the intra-assay variation coefficient for isolated mitochondria from the brain and liver. For both tissues, intra-assay %CV for ATP content and production was between 19.0 and 19.7%, respectively. To obtain the interassay variation coefficient, the variability of the assay from one animal to the next, the average quantitative value for ATP content and production for each tissue was divided into one of three variables (young, old, and caloric restricted) (Table 3). The SD of each group of variables was divided by the respective average from that group and multiplied by 100 to create a percentage (%CV). Hence three variables yielded three individual %CV. These three %CV were then averaged to give the interassay variation coefficient for isolated mitochondria from the brain and liver from one animal to the next. For both tissues, interassay %CV for ATP content and production yielded values of 53.5 and 49.6%, respectively.
It has been proposed that as we age, an accumulation of mitochondrial damage from reactive oxygen species leads to a degradation in the efficiency of the respiratory chain and hence a decline in ATP production (8-10, 47). Moreover, most studies have focused on the activities of mitochondrial matrix enzymes and respiratory enzyme complexes to determine mitochondrial viability and function. Few studies have determined the actual rate of ATP production in isolated mitochondria in vivo. The following ATP determination assay, as well as the preceding methodology, presents a highly sensitive, reproducible method to quantitatively measure in vivo ATP content and rate of ATP production in isolated mitochondria. A limitation with this method hinges on the assumption that the intact mitochondria isolated for these studies are all functional and that any inert, damaged, or unhealthy mitochondria have not been isolated. Recently, we used this method to measure the ATP content and production in both the heart and skeletal muscle of aging and caloric-restricted rats (11) and found a 50% decline in ATP content and the rate of ATP production in skeletal muscle, but not in the heart.
In this paper we describe the stability, inhibition using oligomyocin, and ATP content and production in the liver and the cerebral cortex with age and calorie restriction. This step-by-step approach should allow the easy use for the protocol to measure ATP production in vivo and demonstrate the high sensitivity of this assay. Other studies show that the sensitivity of the luciferin/luciferase reaction has been used to detect trace levels of ATP attributed to low-level bacterial contamination (19). Its sensitivity was apparent in our study showing its ability to detect a 5 nmol ATP standard (Fig. 1B;10 μl of 500 nmol diluted to 1,000 μl total volume). Moreover, when the diluted standard was diluted another 500-fold, there still was a linear response to the reaction between ATP and luciferin/luciferase (see reactions 1 and 2). Hence, it is evident that the sensitivity of the aforementioned assay can detect levels of ATP as low as 10 pmol.
Beyond the sensitivity of the assay, the ATP determination kit was shown to be stable. The luminescent signal of the reaction mixture showed a slight decay of 2.16%/min (Fig. 1A). However, because the quantum signal of the reaction mixture is on the order of ∼10-100 times less than the yield observed for the ATP content and ∼100-1,000 times less than that observed for the rate of ATP production, the decay rate seen for the reaction mixture is negligible. For a 0.5 μmol ATP standard (Fig. 1B), which corresponds to the linear response observed for the ATP content, the linear decay was 1.39%/min. The ATP content was taken as the highest OD reading (usually the 1st or 2nd reading). Thus any decay beyond the first reading would have no effect on its value and would be negligible to the quantitation of the ATP content.
Following the first reading, five more subsequent readings are taken for a total analysis time of 30 s (a total of 6 measurements). The final reading taken at 30 s is utilized in the determination of the rate of ATP synthesis by factoring out the contribution of the signal from the ATP content, the signal from the 2.5 mM ADP (33) in isolation buffer and reaction mixture alone, as well as the decay of the bioluminescent signal associated with it. Hence the determination of the rate of ATP production is exclusive of these aforementioned parameters and is indicative of the production of ATP. The decay rate of a 25.0 μmol ATP standard (Fig. 1C) yields a quantum signal that is parallel to the intensity observed for the production of ATP in isolated mitochondria. It is assumed that the decay rate defined by Eq. 1 is concurrent in the analysis of ATP synthesis in isolated mitochondria and digresses from linearity due to the high concentration. Consequently, this is where the intrinsic deviations from the Beer-Lambert law take effect.
This decay is probably due to combination of different factors such as deviations in absorbance coefficients at higher concentrations due to electrostatic interactions of luciferyl adenylate on the luciferase substrate (see reaction 1) in close proximity, scattering of light due to the relatively high concentration of ATP in the sample, fluorescence or phosphorescence of the sample during the reaction (see reactions 1 and 2), changes in the refractive index due to analyte concentration, and/or shifts in chemical equilibrium as a function of increased concentration of ATP. Applying Eq. 1 to a 30-s analysis, the luminescent sample would decay by 13.4%. While the average values are given for the rate of ATP production as well as the SD about the mean, it can be assumed that there is an intrinsic variation contemporaneous with the decay rate seen in the higher concentration standards.
We tested oligomycin, a strong inhibitor of oxidative phosphorylation, in its ability to attach to and inhibit the F1-ATPase complex. Oligomycin functions by binding to the stalk region of the F0F1-ATPase enzyme and inhibiting the proton pump of the mitochondrial inner membrane, thus impeding the production of ATP. Various concentrations of oligomycin were able to significantly inhibit the production of ATP by 34% (1 mM) and 83% (5 mM) (Fig. 2). Hence this demonstrates the resilience of the F0F1-ATPase enzyme to function even after incubation with a relatively high concentration of oligomycin, an inhibitor of oxidative phosphorylation.
To determine the precision and variability of the assay, the intra- and interassay coefficients of variation were determined (Table 3). The intra-assay %CV, an indicator of precision from one measurement to the next on the same tissue, yielded values of 19.0% for the ATP content and 19.7% for the rate of ATP production for both tissues, brain and liver. While values of 10% are considered ideal, the intra-assay %CV determined for this methodology is good and is indicative of the reason measurements were run in triplicate and then averaged. The interassay %CV, an indication of variability of the assay from one animal to the next, gave values of 53.5 and 49.6% for the measurements of ATP content and rate of ATP production, respectively. This interassay %CV shows the inherent variation seen in vivo for biological markers as shown by others (3, 17, 23).
Neurological diseases and diseases associated with age, such as Alzheimer's (25, 46), Parkinson's (1, 16, 34-36, 39, 49), and Huntington's (6, 22), have all been partly attributed to decreases in the function of respiratory chain complexes and the assumption that there is a lack of ATP production and content. A deficiency in key energy-metabolizing enzymes, such as cytochrome c oxidase (complex IV), could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process. Moreover, liver diseases, such as Wilson disease, are associated with frequent, diverse, and early deletions of mitochondrial DNA (mtDNA), which could lead to altered energy production, because respiratory complexes contain subunits encoded in mtDNA (24, 50). The occurrence of specific base pair deletions are consistent with liver mitochondrial respiratory function decline with age (50). Surprisingly, little is known as to what the effects of a decrease in specific respiratory chain complex activities are on ATP production in vivo in isolated mitochondria from disease pathologies and in the normal aging brain.
Previously, Davey et al. (7, 8) showed that titration of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited before causing impairment of mitochondrial energy metabolism. Using rat brain mitochondria of synaptic origin, the activities of complexes I, III, and IV of the respiratory chain were decreased by 25, 80, and 70%, respectively, before major changes in the rates of oxygen consumption and ATP synthesis were observed (7, 8). Respiratory chain complexes from mitochondria of nonsynaptic origin showed similar resilience to inhibitors (72, 70, and 60%), respectively, before ATP production was reduced (7, 8). From our data and others (7, 8), it appears that electron transport chain complexes are more resilient to reactive oxygen species than previously thought and that any inhibition in ATP production would be a result of a severe impairment to one or more of these complexes (7, 8, 30, 34, 36). Therefore, acute reduction in ATP content and rate of production in mitochondria is only due to severe damage to the respiratory complexes, possibly leading to the aforementioned age-related disease states (7, 8, 24, 45, 50).
In vivo, in the brains of normally aging humans and rats there is a disparity of results regarding a correlation between respiratory chain complex activities and energy production. Ojaimi et al. (28) show that there is a significant decrease in cytochrome c oxidase activity in several brain regions (frontal cortex, superior temporal cortex, cerebellum, and putamen) with age in humans. However, this study is far from conclusive, because only 12 human subjects were studied, while there were only 2 younger subjects used for statistical comparison (26 and 36 yr of age). Furthermore, a study conducted by Hansford's group (12) suggests that there is little evidence of a decrease in respiratory enzyme complex activities. In young 6-mo-old Wistar rats, deletion-containing mtDNA were present at low levels (∼0.0003%) in the striatum, hippocampus, cerebral cortex, and cerebellum, and mtDNA levels increased 25-, 7-, 3-, and 2-fold in these brain regions of 22- to 23-mo-old rats, respectively. Importantly, the activities of mitochondrial respiratory chain complexes I, III, IV, and V, the mitochondrial ATPase, each of which contains subunits encoded in mtDNA, showed no age-related decrements in activity in any of the brain regions (12). In contrast, another study shows that there is a moderate decline in activities of mitochondrial enzymes NADH-cytochrome c reductase, cytochrome oxidase, and citrate synthase, by ∼14-58% in 72-wk-old mice (26). Nicoletti et al. (27) examined contents of subunits cytochrome c oxidase and of subunits of F0F1 ATP synthase from cerebral cortex of rats of different ages (2, 6, 12, 18, 24, and 26 mo). All cytochrome c oxidase subunits showed an age-related increase from 2-mo-old rats up to 24 mo with a small decrease at the oldest age (26 mo). The same pattern of age-dependent changes was observed for γ-ATP synthase, while the α- and β-subunits increased progressively up to 26 mo. Thus, while mtDNA deletions increase with age, they appear to have no significant effect on capacity for oxidative phosphorylation of distinct brain regions, and there are no previous studies that suggest a failure in energy production with age in the brain. In our study using very old Fischer-344 rats (27-28 mo old; mean life span is 24 mo old) we found no decline in ATP production in the brain and liver, suggesting that the selective deletions and changes to respiratory complexes that may occur with age have no overall effect on the capacity of these tissues to produce energy under basal conditions.
Caloric restriction is one of the only proven, preventative methods for inhibiting the effects of age and extending life span. Moreover, caloric restriction has been shown to decrease the production of reactive oxygen species and reduce oxidative stress with increased age (15, 20, 21, 31, 40, 41). Hence, an assumption could be that lifelong caloric restriction should lead to a higher concentration as well as a higher rate of production of ATP within the isolated mitochondria compared with ad libitum-fed animals. This experimental model would examine the hypothesis that the rate of aging may depend on the production of cellular ATP. However, in the cerebral cortex of the brain, no change was observed with age or with caloric restriction on the amount of ATP present or on the rate of production of ATP in the brain. In the liver, neither age nor caloric restriction had any significant effect on the amount of ATP present or on the rate of ATP production.
Recently, others have investigated the effects of long-term caloric restriction on extracts from brain mitochondrial fractions and mitochondria isolated from the heart and skeletal muscle. Olgun et al. (29) found no significant difference in the protein levels of complexes I and III between ad libitum-fed and long-term caloric-restricted mice (alternative day feeding). Complex IV activity increased by 8% with caloric restriction, but this change was not statistically significant. Furthermore, recent studies by Sreekumar et al. (42) and Drew et al. (11) found that caloric restriction did not have any major impact on ATP production and/or mitochondrial function in muscle and heart.
In conclusion, this study demonstrates that the following methodology provides a sensitive means to quantitatively measure ATP content in addition to the rate of synthesis in isolated mitochondria. Moreover, age and caloric restriction had no major effects on energy production in the mitochondria isolated from the brain and the liver. The relationship between mitochondrial complex damage-inhibition and ATP synthesis has been recently reviewed by Rossignol et al. (32). They describe three “mitochondrial threshold effects.” The first shows that, in most cases, phenotypic manifestation of a genetic defect (mtDNA deletion) occurs only when a threshold level is exceeded, named the “phenotypic threshold effect.” Second, considerable inhibition of the activity of a respiratory chain complex can occur, up to a critical value (often 50%), without affecting the rate of mitochondrial respiration or ATP synthesis: the “biochemical threshold effect” (32). Third, quantitative analysis of the effects of various mutations in mtDNA on the rate of mitochondrial protein synthesis has revealed the existence of a “translational threshold effect” (32). Future studies should develop methods to determine mitochondrial function in intact tissues to reach firmer conclusions regarding the hypothesis that a lack in energy production is a major contributor to disease and aging. Moreover, broader studies need to be proposed (2, 4, 8, 12, 24 mo of calorie restriction) that could shed light on transient changes with caloric restriction. The lack of differences noted between old ad libitum-fed and old calorie-restricted rats could be suggestive of the compensatory ability of the organism. Moreover, little is known regarding mitochondrial turnover and mitochondrial biogenesis with age and the effects of caloric restriction. It is likely that severely damaged mitochondria are rapidly recognized by lysosomes and degraded while mitochondrial biogenesis provides newly remodeled mitochondria (13, 18, 44).
The National Institute of Aging supported this research (Grants AG-17994, AG-10485, and AG-21042), as well as American Heart Association, Florida-Puerto Rico Affiliate Grants 30334B and 0225194B.
We thank Dr. C. Selman, S. Phaneuf, and A. Hiona for input and for critical reading of the manuscript.
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.
- Copyright © 2003 the American Physiological Society