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Department of Medical Physiology (J.E.P., I.C.C.), School of Medicine, University Universidad San Pablo-CEU, 28668 Madrid, Spain; Department of Medical Physiology (J.E.P., M.G.-F., J.R., S.G.-B., I.C.C.), School of Medicine, University of Málaga, 29071 Málaga, Spain; and Department of Internal Medicine (J.M.), Liver Unit, University of Córdoba, 14080 Córdoba, Spain
Address all correspondence and requests for reprints to: Inma Castilla Cortazar, M.D., Department of Medical Physiology, School of Medicine, University CEU-Universidad San Pablo, Boadilla del Monte, 28668, Madrid, Spain. E-mail: iccortazar{at}uma.es.
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Abstract |
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Introduction |
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We have recently reported that the exogenous administration of low doses of IGF-I restores IGF-I circulating levels and some age-related changes, improving glucose and lipid metabolisms, increasing testosterone levels and serum total antioxidant capability, and reducing oxidative damage in the brain and liver associated with a normalization of antioxidant enzyme activities and mitochondrial membrane potential (MMP) (4). From these results we suggested that aging seems to be an unrecognized condition of "IGF-I deficiency." The best-known condition of "IGF-I deficiency" is Laron’s dwarfism (5), characterized by an absence of GH receptors in the liver. Another condition of IGF-I deficiency is liver cirrhosis. In cirrhosis the reduction of receptors for GH in hepatocytes and the diminished ability of the hepatic parenchyma to synthesize cause a progressive decrease in serum IGF-I levels (6). We have shown previously that short courses of treatment with low doses of IGF-I in rats with carbon tetrachloride-induced cirrhosis induced many systemic beneficial effects, and showed hepatoprotective and antioxidant properties, including mitochondrial protection (7, 8, 9, 10, 11, 12).
The reported cytoprotective (neuroprotective and hepatoprotective) activity of IGF-I in aging rats (4) could also be related to mechanisms of mitochondrial protection. In fact, in the study by García-Fernández et al. (4), untreated aging animals showed a depletion of MMP with a significant reduction of ATP synthesis, which IGF-I replacement therapy was able to correct to the normal values of young rats.
Mitochondria are particularly sensitive to damage induced by reactive oxygen species (ROS) in the pathogenesis of disease and aging (13, 14). Normal mitochondrial function is a critical place in maintaining cellular homeostasis because mitochondria produce ATP and are the major intracellular source of free radicals. Cellular dysfunctions induced by intracellular or extracellular insults converge on mitochondria and induce a sudden increase in permeability on the inner mitochondrial membrane, the so-called mitochondrial membrane permeability transition (MMPT). MMPT is caused by the opening of pores in the inner mitochondrial membrane, matrix swelling, and outer membrane rupture. The MMPT is an endpoint to initiate cell death because the pore opening together with the release of mitochondrial cytochrome c activates the apoptotic pathway of caspases. Cellular targets of the effector caspases include endonucleases and cytoskeletal proteins (15). Adenine nucleotide translocator (ANT) catalyzes the exchange of ATP and ADP between the mitochondria and the cytosol. It has also been shown to be a significant mediator of the basal proton leak in mitochondria and one of the most sensitive points for pore opening (16).
Understanding that mitochondria are one of the most important cellular targets of IGF-I (4, 11, 12), the aims of the present study were to characterize the mitochondrial dysfunction in aging rats and to investigate the effect of IGF-I therapy on damaged mitochondria leading to cellular protection. With these aims, we have extended this study to analyze the effect of IGF-I on mitochondrial function parameters, such as MMP, oxygen consumption, intramitochondrial free radical production, proton leak rates, cytochrome c oxidase and ATPase activities, inhibition of ANT by atractyloside (Atr), intramitochondrial oxidative damage, and intramitochondrial antioxidant enzyme activities (16, 17, 18, 19, 20, 21, 22, 23). All of these were assessed in isolated hepatic mitochondria, and caspase 3 activation and caspase 9 were determined in liver homogenates in the following experimental groups: young healthy controls (yCO) (17 wk old, n = 6); untreated old rats (O group) (103 wk old, n = 6); and aging rats (103 wk old) treated with IGF-I (O + IGF-I) during 1 month (2.25 µg IGF-I/100 g body wt–1·d–1, n = 6).
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Materials and Methods |
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Both food (standard semipurified diet for rodents; B.K. Universal, Sant Vicent del Horts, Spain) and water were given ad libitum. Rats were housed in cages placed in a room with a 12-h light, 12-h dark cycle, and constant humidity and temperature (20 C).
In the morning of the 31st day, rats were killed by decapitation, and the liver was dissected. Fresh liver was used to isolate mitochondria and to perform mitochondrial function tests using flow cytometry (4, 11). Samples were obtained simultaneously from young, old-untreated rats, and old rats treated with IGF-I to have paired data.
Isolation of liver mitochondria
Liver mitochondrial fraction was prepared according to the method described by Schneider and Hogeboom with modifications (11, 17, 42). Liver samples were homogenized (1:10 wt/vol) in an ice-cold isolation buffer containing sucrose 0.25 M and 0.1% BSA buffered (pH 7.4) with Tris-HCl 10 mM, and the isolation medium was identical without BSA or EDTA. The protein concentration was measured using the Biuret method. The homogenate was centrifuged at 800 x g for 10 min. The resulting supernatant was centrifuged at 8500 x g for 10 min. The supernatant was discarded, and the pellet was diluted in cold isolation buffer and centrifuged at 8500 x g for 10 min three times. The final mitochondria pellet was resuspended in a minimal volume, and aliquots were stored at –80 C until use in enzyme assays. All procedures were conducted at 4 C.
Unless otherwise indicated, the standard incubation medium had the following composition: 100 mM NaCl, 5 mM sodium-potassium phosphate buffer (pH 7.4), 10 mM Tris-HCl buffer (pH 7.4), and 10 mM MgCl2. The respiratory substrates used were 5 mM potassium glutamate plus 2.5 mM potassium malate and 5.0 mM potassium succinate plus 4 µM rotenone.
Oxygen consumption
Oxygen consumption was measured using a Clark-type electrode (Hansatech Instruments Ltd., using software OXIGRAPH version 1.10; Norfolk, UK) in a 2-ml glass chamber equipped with magnetic stirring. The reaction was started by the addition of 6 mg mitochondrial protein to 2 ml standard medium containing rotenone 5 µM, oligomycin 1.3 mM, nigericin 100 pmol/mg protein, succinate 10 mM, ADP 200 µM, and glutamate/malate 5/2.5 mM. Finally, it was stabilized for 1 min at 30 C. Respiration rates are given in nAtom-gram oxygen/mg·min. Phosphorylating respiration (state 3) was initiated by addition of 200 nmol ADP/mg protein. Phosphorylation efficiency (ADP/O ratio) was calculated from the added amount of ADP and total amount of oxygen consumed during state 3. The state 4 is obtained with all substrates but ADP.
The ratio between state 3 rate and state 4 rate is called the respiratory control ratio (RCR), and indicates the tightness of the coupling between respiration and phosphorylation. With isolated mitochondria the coupling is not perfect, probably as a result of mechanical damage during the isolation procedure. Typical RCR values range from three to 10, varying with the substrate and the quality of the preparation. Coupling is thought to be better in vivo but may still not achieve 100%.
Flow cytometry analysis
Gated mitochondrial population was chosen by flow cytometry, based on forward scatter and side scatter within mitochondria samples, after obtaining one clear mitochondria population.
Mitochondrial transmembrane electrical potential.
MMP (
) was measured by the lipophilic cationic fluorescent probe Rh-123 (Molecular Probes Inc., Eugene, OR), a fluorescent derivative of uncharged dihydroRh-123, according to previous studies (4, 11, 17). A mitochondrial suspension (50 µg/ml) was incubated with the same respiratory substrates used in oxygen uptake for 1 min at room temperature, and after adding Rh-123 (260 nM) and incubating it for another minute. After incubation, suspensions were immediately analyzed by flow cytometry. The values of the fluorescence (FL) substrates were normalized to the value obtained with the uncoupler carbonylcyamide-m-chlorophenylhydrazone.
Rate of intramitochondrial free radical generation.
The rate of ROS generation from mitochondria was measured after the formation of Rh-123 using the cytometry method performed by O’Connor (17) with a small modification. A mitochondrial suspension (100 µg/ml) was incubated with 0.82 nM dihydro Rh-123 and 7 U/ml horseradish peroxidase for 5 min at room temperature. The values of the FL substrates were normalized to the value obtained without peroxidase and adding the uncoupler CCCP. H2O2 (1 mM) was used with the positive control.
After incubation, the suspensions were immediately analyzed. Gated mitochondrial population was chosen by flow cytometry, based on forward scatter and side scatter within mitochondria samples.
Cytofluorometric analysis was performed using a flow cytometer EPICS XL (Beckman Coulter, Inc., Fullerton, CA) equipped with a single 488-nm argon laser (15 mW). Green FL was detected with a wide-band filter for Rh-123 centered in 525 ± 20 nm (FL1). A standard cytogram based on the measurement of right angle scatter vs. forward angle scatter was defined to eliminate cellular debris and aggregates. A minimum of 10,000 mitochondria per sample was acquired in list mode and analyzed with System II version 3.0 software (Beckman Coulter).
Activities of mitochondrial complexes
Mitochondrial suspensions were thawed and diluted with potassium phosphate. Activities of the respiratory chain enzymes were measured at 37 C in Cobas Mira (ABXMicro, Mannheim, Germany).
Measurements of cytochrome oxidase activity.
Cytochrome oxidase activity was measured according to the method described by Cortese et al. (23). Mitochondria were resuspended in the medium containing (in mM) 220 mannitol, 70 sucrose, 2.5 K2HPO4, 2.5 MgCl2, and 0.5 EDTA. Antimycin A was then added to block mitochondrial respiration through complex III. Reaction was started by adding ascorbate/N,N,N’,N’-tetramethyl-p-phenylenediamine as an electron donor.
Complex V, ATPase (EC 3.6.1.34.)
The activity was assayed by coupling the reaction to the pyruvate kinase and lactate dehydrogenase systems, and measuring reduced nicotinamide adenine dinucleotide (NADH) oxidation at 340 nm. The assay system contained Tris-HCl buffer 65 mM (pH 7.5), sucrose 300 mM, MgCl2 4.75 mM, ATP 4 mM, NADH 0.4 mM, phosphoenolpyruvate 0.6 mM, potassium cyanide 5 mM, pyruvate kinase 700 U/ml, and lactate dehydrogenase 1000 U/ml.
Assessment of "proton leak": the relationship between respiration rate and MMP ()
Mitochondrial proton leak was calculated from respiration rates and MMP expressing the ratio of protons for each oxygen atom consumed (18, 19, 20, 21). The rate of proton leak across the inner mitochondrial membrane is a function of the driving force (membrane potential) and increases disproportionately with membrane potential.
Titration of membrane potential and state 4 oxygen consumption by respiratory inhibitors were performed simultaneously in separate vessels at 30 C. Nigericin was added to collapse the pH difference across the mitochondrial inner membrane and, thus, had the value of the proton motive force (p). Reactions were started by the addition of 3 mg mitochondrial protein/ml standard medium containing also 3 mM rotenone, 1.3 mM oligomycin, nigericin (100 pmol/mg protein), and 5 mM succinate. The addition of inhibitors was begun when the maximum value of the potential became stable (after 
2–3 min). When succinate was used as the substrate, the titration was performed with malonate (K/salt) from 0–13 mM; at the end of each membrane potential trace, the zero point was determined by addition of CCCP 1 mM. Rates of respiration during the titration with inhibitors were measured with a Clarke-type oxygen electrode, and membrane potential with a flow cytometer simultaneously with the measurements of membrane potential.
Inhibition of ANT by Atr
To establish the optimal concentration of Atr (Calbiochem-Novabiochem, San Diego, CA) needed for ANT inhibition, the efficiency of Atr was first examined in its classical role, i.e. for its ability to inhibit oxidative phosphorylation. For analysis, increasing Atr concentrations (50–200 pmol/mg mitochondrial protein) were used until complete inhibition of oxygen consumption was obtained (22).
Oxidative damage and total antioxidant status (TAS) in isolated mitochondria
Lipid hydroperoxides (LOOHs) were assessed in isolated mitochondria as previously described by Arab and Steghens (25), and adapted for Cobas Mira (600-nm wavelength) and mitochondria suspensions. Briefly, orange xylenol (180 µl–167 µM) was added to 25 µl sample. The first optic reading was obtained before the addition of iron gluconate (45 µl-833 µM). LOOH was calculated using a standard curve of tert-butyl hydroperoxide, and LOOH levels were expressed as nmol/mg mitochondrial protein. Intraassay and interassay coefficients were 3 and 8%, respectively.
TAS, as total enzymatic and nonenzymatic antioxidant capability, was evaluated in isolated mitochondria by a colorimetric assay (Randox Laboratories Ltd., Ardmore, Crumlin, UK) using the following principle: 2,2'-azino-di-(‘3-ethylbenzthiazoline sulfonate) was incubated with a peroxidase (metmyoglobin) and H2O2 to produce the radical cation 2,2'-azino-di-(‘3-ethylbenzthiazoline sulfonate) · +. This has a relatively stable blue-green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree that is proportional to their concentration (26, 27).
Activities of antioxidant enzymes in isolated mitochondria
Activities of superoxide dismutase (SOD) (EC 1. 15. 1. 1.), glutathione reductase (GRD) (EC 1.6.4.2), and glutathione peroxidase (GSHPx) (EC 1.11.1.9.) were measured in liver mitochondria. Antioxidant activities were determined at 37 C using a commercial kit (Ransod; Randox Laboratories) and an autoanalyzer (Cobas Mira; Roche Diagnostic System, Basel, Switzerland).
Western blots for caspase 3 and caspase 9 in liver homogenates
Frozen livers (1 g) were homogenized (Ultra-turrax T25; Janke & Kunkel IKA-Laboratory, Staufen, Germany) in lysis solution [1% sodium dodecyl sulfate, Tris-HCl 10 mM, EDTA 50 mM, phenylmethylsulfonylfluoride 1 mM, aprotinin 1 µg/ml, and leupeptin 1 µg/ml (pH 7.4)] at 4 C for 10 min, transferred to Eppendorf tubes, and centrifuged at 20,800 x g at 4 C for 5 min. Proteins (100 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose. The membrane for measuring caspase 3 activation was incubated with anticaspase 3 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as primary antibodies and antirabbit-IgG-alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) as secondary antibodies using 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride as alkaline phosphatase substrate.
Caspase 9 quantization followed the same procedure but using its specific antibody, anticaspase 9 (Santa Cruz Biotechnology).
Statistical analysis
Data are expressed as means ± SEM. Statistical significance was estimated with the paired or unpaired t test as appropriate. A P value less than 0.05 was considered significant. All analyses were performed using the SPSS version 10.0 (SPSS, Inc., Chicago, IL) statistical package.
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Results |
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Table 1
summarizes the MMP values, expressed as arbitrary units (AU), in the three experimental groups. According to preliminary results (4), a reduction of MMP was observed in untreated aging rats compared with young controls, which IGF-I replacement therapy was able to restore to similar values to those found in young controls.
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shows oxygen consumption under different conditions and RCRs in mitochondria from the three experimental groups.
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and in a preliminary study (4). In addition, the ratio ADP to O expressing oxidative phosphorylation as ATP produced by oxygen molecule consumed, was significantly reduced in mitochondria from O group (P < 0.05 vs. yCO and O + IGF-I groups), whereas old rats treated with low doses of IGF-I showed similar values to those found in young controls.
No significant differences were found among the three experimental groups in RCRs (state 3 to state 4).
Proton leak rates.
The rate of proton leak across the inner mitochondrial membrane is a function of the driving force (membrane potential) and increases disproportionately with membrane potential (18, 19, 20, 21). Proton leak rates express proton "escape" into mitochondrial matrix contributing to dissipation of the MMP in pathological conditions.
Figure 1 summarizes proton leak curves in the three experimental groups, expressed by oxygen consumption (nAgO/mg·min–1) at a given MMP in state 4 (without ADP). Mitochondria from O needed to consume increased oxygen compared with young controls to reach the same MMP values. Figure 1 showed that the proton leak curve of mitochondria from old animals treated with IGF-I was similar to the curve corresponding to young controls.
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As shown in Fig. 2, ATPase activity (expressed as µmol ATP per mg protein) was significantly reduced in untreated aging rats. However, there were no significant differences between yCO and O + IGF-I groups in complex V activity, according to preliminary data (4).
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shows the intramitochondrial ROS production in isolated mitochondria from the three experimental groups in complex I (Fig. 3 A) using glutamate/malate as substrates and complex III (Fig. 3 B) with succinate plus antimycin as substrates. In both places, mitochondria from untreated aging rats showed a significant increase of ROS generation compared with mitochondria from young controls and O + IGF-I.
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, Atr blocked oxygen consumption in mitochondria from young controls showing a normal inhibition of ANT. Full inhibition of the oxygen consumption induced by addition of ADP was obtained in liver mitochondria from young controls at a concentration of 150 pmol/mg Atr.
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). Interestingly, Atr inhibition was similar to young controls in mitochondria from O + IGF-I showing a normal inhibition of ANT. In this group (O + IGF-I) as shown in Fig. 4, full inhibition of the oxygen consumption induced by addition of ADP was obtained at a concentration of 200 pmol/mg Atr.
Mitochondrial oxidative damage, TAS, and intramito- chondrial antioxidant enzyme activities in isolated liver mitochondria
Table 3 summarizes intramitochondrial oxidative damage, using LOOHs as markers, and total antioxidant capability of isolated mitochondria (26, 27) as well as the activities of mitochondrial antioxidant enzymes.
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However, GSHPx activity was significantly increased in both groups of old rats. In contrast, SOD activity was reduced in old rats, particularly in mitochondria from old rats treated with IGF-I. No significant differences were found between groups in GRD activity.
Measurements of caspase 3 and caspase 9 activation.
Western blot for fragment 17 of caspase 3 showed a significant increase of caspase 3 activation in untreated aging rats compared with young controls (Fig. 5). However, a notable reduction in the expression of the active fragment of caspase 3 was observed in old animals treated with IGF-I.
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; P < 0.06). Again, IGF-I treatment induced a significant reduction of this parameter in old rats. |
Discussion |
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In fact, the administration of low doses of IGF-I restored IGF-I circulating levels, and were able to exert many beneficial effects on age related-changes improving testosterone levels, insulin resistance, lipid metabolism, and oxidative damage on brain and liver (4). We suggested that the described cytoprotection (neuroprotection and hepatoprotection) induced by IGF-I replacement therapy could be related to mitochondrial protection mechanisms. Thus, the present study was designed to analyze the effect of IGF-I on mitochondrial function leading to cellular protection. The mechanisms of IGF-I action are not completely understood, but data in this paper give some insight.
Compared with healthy young controls, untreated aging rats showed an increase of mitochondrial oxidative damage with a mitochondrial dysfunction characterized by depletion of membrane potential with increased proton leak and intramitochondrial free radical production, and a significant reduction of ATPase activity. In addition, mitochondrial respiration from untreated aging rats was Atr insensitive, showing that ANT was uncoupled, probably due to peroxidation of thiol groups (28, 29). ANT has been shown to be one of the most sensitive places for pore opening. Consistent with these results, untreated aging rats showed a significant overexpression of the active fragment of caspase 3 and caspase 9. All of these findings contribute to characterize better the mitochondrial dysfunction associated with aging in this experimental model in rats. Results in this paper are in agreement with a significant amount of evidence that considers oxidative damage as one of the predominant mechanisms of cellular and tissular damage in aging. The ensuing oxidative stress leads to lipid peroxidation, mitochondrial dysfunction, and depletion of ATP (13, 27, 30, 31, 32, 33). In aging, the selectively diminished activities of complexes I and IV have also been reported (32). In addition, it has been described that the ratio of ATP over oxygen consumed is reduced in aging, and this is associated with insulin resistance (31). Accordingly, we have recently reported that aging rats with mitochondrial dysfunction showed insulin resistance (4).
The major finding of this work was that mitochondrial dysfunction leading to apoptosis (activation of caspases) was improved by IGF-I therapy. In fact, IGF-I replacement therapy normalized mitochondrial oxidative damage and mitochondrial function (MMP and ATP synthesis), reducing intramitochondrial free radical generation, proton leak rates, and the vulnerability for pore opening in ANT, which was associated with a reduction of caspase 3 and 9 activation compared with O group.
The observed reduction of MMP with an increased generation of free radicals in aging rats suggests that oxygen is wasted by damaged mitochondria producing H2O2 instead of a normal proton gradient, the driving force of ATP synthesis through the proton pumping F1F0 ATP synthase (ATP synthase). In addition, proton leak rates were altered in aging rats. The rate of proton leak across the inner mitochondrial membrane increases disproportionately with membrane potential (13). Recently, ANT has been a significant mediator of the basal proton leak in mitochondria (21). In damaged mitochondria, peroxidation of ANT (or uncoupling proteins) facilitates the escape of protons into the mitochondrial matrix contributing to the MMP dissipation (16, 18, 19, 20).
Another point that deserves particular mention is that ANT has been one of the most sensitive places for mitochondrial pore opening (16, 34) for the release of cytochrome c activating caspases in cytosol (23). ANT contains a thiol group of a cysteine residues (Cys56) that are easily oxidized by ROS (16). In physiological conditions, Atr is able to compete with ADP in ANT blocking mitochondrial respiration. As was shown in Fig. 4, Atr blocked oxygen consumption induced by the addition of ADP in mitochondria from young controls showing a normal inhibition of ANT. Peroxidation of ANT-thiol groups causes an uncoupling of ANT. In this condition of oxidative mitochondrial damage, respiration is Atr insensitive. Thus, in this study Atr did not inhibit respiration in mitochondria from untreated aging rats (Fig. 4). This result is in agreement with other authors who have reported that aging enhances the activation of the permeability transition pore in mitochondria (34).
Of interest in the present work, IGF-I therapy reduced oxidative mitochondrial damage, correcting all these parameters of mitochondrial dysfunction, and resulting in a reduction of caspase 3 and 9 activation compared with O group. Together, these data suggest an extramitochondrial protection of mitochondria, which is not fully understood. Previously, we reported that low doses of IGF-I restored the expression of several protease inhibitors such us 
-1-antichymotripsin, the serine protease inhibitor 2 in cirrhotic rats (35), which could contribute to the described mitochondrial protection. In agreement with these results, it has been reported that IGF-I differentially regulates Bcl-xL and Bax, and confers myocardial protection in the rat heart (36).
In recent years, mitochondria have been recognized as regulators of cell death (13, 15, 22, 23). Intracellular or extracellular insults converge on mitochondria, inducing the so-called MMPT. The MMPT is an endpoint to initiate cell death and a putative target for cellular protection because MMPT and the release of mitochondrial cytochrome c activate the apoptotic pathway by which initiator caspases (i.e. caspases 8 and 9) are converted to their active forms, which in turn activate downstream effector caspases (i.e. caspases 3, 6, and 7) (15). Finally, cellular targets of the effector caspases include endonucleases and cytoskeletal proteins (13, 15).
These results show that IGF-I induces cell resistance to apoptosis by oxidative stress through mitochondrial protection. Mitochondria seem to be one of the most important cellular targets of IGF-I actions. Likewise, our data are in agreement with the observation that the effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-I (34). Accordingly, it has been reported that IGF-I inhibited the reduction of MMP, cytochrome c release, caspase 3 activity, and apoptosis in several cell lines and experimental procedures (38, 39, 40, 41).
The described improvement of mitochondrial function in aging rats by IGF-I therapy resulted in an increment of ATP synthesis. Interestingly, several beneficial metabolic effects of IGF-I in aging (4) could be related to an increment of ATP availability after IGF-I therapy.
In conclusion, results in this paper show that the cytoprotective effect of IGF-I is closely related to a mitochondrial protection, leading to the reduction of intramitochondrial free radical production, oxidative damage, and apoptosis, and to increased ATP production. This work provides new evidence of the beneficial effect of IGF-I replacement therapy in aging.
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Acknowledgments |
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Footnotes |
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First Published Online February 14, 2008
Abbreviations: ANT, Adenine nucleotide translocator; Atr, atractyloside; AU, arbitrary unit; EC, Enzyme Commission of the International Union of Biochemistry; FL, fluorescence; GRD, glutathione reductase; GSHPx, glutathione peroxidase; LOOH, lipid hydroperoxide; MMP, mitochondrial membrane potential; MMPT, mitochondrial membrane permeability transition; ns, not significant; O, untreated old rat; O + IGF-I, aging rats treated with IGF-I; RCR, respiratory control ratio; ROS, reactive oxygen species; SOD, superoxide dismutase; TAS, total antioxidant status; yCO, young healthy control.
Received November 13, 2007.
Accepted for publication February 6, 2008.
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