Aging skeletal muscle mitochondria in the rat: decreased uncoupling protein-3 content

Janos Kerner1,3, Peter J. Turkaly2, Paul E. Minkler3, and Charles L. Hoppel2,3

Departments of 1 Nutrition, 2 Pharmacology and Medicine, Case Western Reserve University, and 3 Veterans Affairs Medical Research Center, Geriatric Research, Education and Clinical Center, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to discern the cellular mechanism(s) that contributes to the age-associated decrease in skeletal muscle aerobic capacity. Skeletal muscle mitochondrial content, a parameter of oxidative capacity, was significantly lower (25 and 20% calculated on the basis of citrate synthase and succinate dehydrogenase activities, respectively) in 24-mo-old Fischer 344 rats compared with 6-mo-old adult rats. Mitochondria isolated from skeletal muscle of both age groups had identical state 3 (ADP-stimulated) and ADP-stimulated maximal respiratory rates and phosphorylation potential (ADP-to-O ratios) with both nonlipid and lipid substrates. In contrast, mitochondria from 24-mo-old rats displayed significantly lower state 4 (ADP-limited) respiratory rates and, consequently, higher respiratory control ratios. Consistent with the tighter coupling, there was a 68% reduction in uncoupling protein-3 (UCP-3) abundance in mitochondria from elderly compared with adult rats. Congruent with the respiratory studies, there was no age-associated decrease in carnitine palmitoyltransferase I and carnitine palmitoyltransferase II activities in isolated skeletal muscle mitochondria. However, there was a small, significant decrease in tissue total carnitine content. It is concluded that the in vivo observed decrease in skeletal muscle aerobic capacity with advanced age is a consequence of the decreased mitochondrial density. On the basis of the dramatic reduction of UCP-3 content associated with decreased state 4 respiration of skeletal muscle mitochondria from elderly rats, we propose that an increased free radical production might contribute to the metabolic compromise in aging.

aging; fuel utilization; carnitine palmitoyltransferase; carnitine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AGING is associated with a progressive decline in muscle performance, characterized by decreased muscle strength and endurance capacity in both humans (30) and animals (6, 50). Although the reduction in muscle size could account for much of the reduction in muscle strength (see review in Ref. 42), the mechanism(s) underlying the reduced aerobic capacity is less clear. When animal models of aging have been used, a decrease of mitochondrial oxidative function as a cause of reduced aerobic capacity has been implicated by studies that show decreased oxidative enzyme activities in skeletal muscle homogenates (7, 19, 20, 48) as well as in isolated mitochondria (13, 49, 50). However, these findings are not supported in other studies, where no age-related changes in oxidative enzyme activities are found in skeletal muscle homogenates (29, 45, 47, 56). Similarly, conflicting results were obtained in functional studies measuring the respiratory properties of isolated mitochondria, reporting either no changes (4, 16) or a decrease in respiratory rates with some but not with other substrates (9, 19). These discrepant findings with respect to age on skeletal muscle aerobic capacity could in part be explained by the different strains and species used in the studies as well as the different muscles sampled (21).

Recent in vivo studies in humans reported decreased fat oxidation in elderly compared with young adults during exercise at either the same absolute or relative intensity (44). Because lipolytic rates and free fatty acid availability were not rate limiting in the elderly subjects and the decrease in fat oxidation was at the expense of carbohydrate oxidation, the authors concluded that aging selectively affects fatty acid oxidation in skeletal muscle. Although the interpretation of the data presented in Ref. 44 have been questioned (5), several in vitro animal studies have indicated a selective age-associated decrease in mitochondrial fatty acid oxidation. Beyer et al. (4) found a significant decline in palmitoyl-CoA oxidation in skeletal muscle homogenates of elderly rats, whereas the oxidation of palmitoylcarnitine was unaffected. Similarly, a highly significant decrease in palmitate oxidation in skeletal muscle homogenates was found by Cartee and Farrar (7) in contrast to the modest age-associated decreases in mitochondrial enzyme activities. Because the oxidation of palmitoyl-CoA (as well as of palmitate) requires the activity of carnitine palmitoyltransferase I (CPT-I), which catalyzes the first committed and regulated step of overall mitochondrial fatty acid oxidation, the data implicate the entry of activated fatty acids into mitochondria as a potential site of the age-linked decline in fatty acid oxidation. In support of this interpretation are reports of age-associated decreases in CPT-I activity in isolated rat and mouse heart mitochondria (37, 40). Decreased CPT activity has also been reported in skeletal muscle of elderly rats; however, no distinction between CPT-I and CPT-II activity has been made in this study (20). Additionally, there is a decrease in skeletal muscle carnitine content with advancing age in several species (7, 11, 20). As the affinity of skeletal muscle mitochondrial CPT-I for carnitine is low (36), the activity of CPT-I could be further dampened by the decreased carnitine content.

Recent studies on the transcriptional regulation of uncoupling protein-3 (UCP-3), the major isoform in skeletal muscle, suggest a role for this protein in the oxidation of fatty acids. UCP-3 expression is upregulated under conditions of increased fatty acid oxidation, such as acute exercise, lipid infusion, and fasting; the fasting-induced upregulation is reversed by refeeding of a low-fat diet, but not by a high-fat diet. This proposed role of UCP-3 in the regulation of lipids as fuel substrate is further supported by the association between UCP-3 polymorphism and basal lipid oxidation in humans (14 and references therein). Additionally, via increased and/or decreased uncoupling of mitochondrial oxidative phosphorylation, changes in UCP-3 expression could also affect ATP availability, as well as free radical production (10, 18, 31, 55).

The goal of the present work was to examine in a comprehensive manner skeletal muscle mitochondrial oxidative metabolism in the aged rat. We have chosen 6-mo- and 24-mo-old animals for our studies. Our decision was based on data reported by Chen et al. (9) and Rumsey et al. (43), who studied the effect of aging on rat skeletal muscle oxidative capacity by using 3-, 12-, 16-, 20-, 24-, and 28-mo-old (9) and 10-, 18-, 24-, and 30-mo-old animals (43). These authors reported no age-associated decrease in rat skeletal muscle oxidative capacity until the animal ages of 20 and 24 mo, respectively. We have determined the mitochondrial content of mixed skeletal muscle, the yield of mitochondrial protein, oxidative phosphorylation, and the expression of UCP-3 in isolated intact skeletal muscle mitochondria. Additionally, because a selective age-associated decrease in palmitate and palmitoyl-CoA oxidation has been reported in homogenates of rat skeletal muscle (4, 7), we also measured the specific activity of CPT-I and CPT-II and determined the skeletal muscle carnitine content.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male 6- and 24-mo-old Fischer 344 rats were obtained from a colony maintained by the National Institute of Aging (Harlan Sprague Dawley, Indianapolis, IN). The animals, housed in our animal care facility in a temperature- and humidity-controlled room and a 12:12-h light-dark cycle, had free access to food and water until the experiments. On the day of experiments, the animals were weighed and anesthetized with Nembutal (100 mg/kg body wt). After euthanasia, the muscles from both hindlimbs were removed. All animals were used between 8 and 9 AM. The mean body weights were 375 ± 18 and 404 ± 25 g in the adult (6-mo) and elderly (24-mo) groups, respectively.

Chemicals. L-[methyl-14C]carnitine, palmitoyl-L-[methyl-14C]carnitine, palmitoyl- and octanoyl-L-carnitine, and etomoxiryl-CoA were synthesized as referenced earlier (24). Affinity-purified rabbit polyclonal antibodies specific for UCP-3 were purchased from Calbiochem. These antibodies were raised against a synthetic linear peptide (GTGGERKYRGTMD) unique to mouse, rat, and Djungarian hamster UCP-3 (similarity search of the SwissProt database by use of the advanced BLAST system), and the antibodies do not cross-react with UCP-2, UCP-1, or human UCP-3 (Calbiochem Biologics, vol. 24.2). The absence of the above sequence in rat UCP-2 and UCP-1 was further confirmed by sequence alignment (ExPASy proteomics server of the Swiss Institute of Bioinformatics) of rat UCP-3, UCP-2, and UCP-1. Horseradish peroxidase-conjugated protein A was from Bio-Rad, and substrates for enhanced chemiluminescent detection were from Amersham Pharmacia Biotech. All other chemicals were commercially available and purchased in the highest quality available.

Preparation of skeletal muscle mince and homogenates. Skeletal muscles from both hindlimbs were removed and placed in ice-cold isolation medium (in mM: 100 KCl, 50 MOPS, 5 MgSO4, 1 EGTA, and 1 ATP, pH 7.4). The muscle tissue was blotted dry, freed of visible fat and connective tissue, finely minced with scissors, and thoroughly mixed. Minced skeletal muscle (10-11 g) was used for mitochondrial isolation, and the remainder of the mince was quick-frozen in liquid nitrogen and stored at -60°C for later analysis of enzyme activities, total carnitine, and noncollagenous protein (NCP).

Skeletal muscle homogenates (5% wt/vol) in 150 mM NaCl, 10 mM potassium phosphate, and 0.1 mM EGTA for enzyme assays, NCP, and carnitine determination were prepared using a hand-held Polytron homogenizer (setting 4, 2 × 30 s on ice).

Isolation of mitochondria. Total skeletal muscle mitochondria were isolated as in Ref. 25, with slight modifications. After several rinses with isolation medium, the skeletal muscle mince was suspended in 10 volumes (wt/vol) of the same medium and treated with the protease Nagarse (5 mg/g mince) for 10 min on ice with constant stirring. The suspension was homogenized with a loose- and then with a tight-fitting Teflon pestle (four strokes each) and was diluted with an equal volume of isolation medium supplemented with defatted bovine serum albumin (BSA) to 0.2% (wt/vol). Nagarse was removed by centrifugation [10 min at an average of 7,802 g (gav)], and the pellet was resuspended in BSA-supplemented isolation medium (10 ml/g tissue) with a loose-fitting Teflon pestle. The resuspended pellet was centrifuged for 10 min at 225 gav, the supernatant fluid was filtered through two layers of gauze, and the mitochondria were sedimented at 4,750 gav for 10 min. The mitochondria were subjected to two additional washes [5 ml BSA-supplemented isolation medium/g tissue and 2.5 ml of (in mM) 100 KCl, 50 MOPS, and 0.5 EGTA, pH 7.4/g muscle] and finally resuspended with ~1.2 ml of (in mM) 100 KCl, 50 MOPS, and 0.5 EGTA, pH 7.4.

Assays. CPT-I was measured by the modified forward assay on freshly isolated mitochondria described in detail previously (24) with the following minor modifications. The molar ratio of palmitoyl-CoA to BSA was increased from 1.0 to 3.4 by decreasing the BSA concentration in the assay to 0.1%, and etomoxiryl-CoA rather than malonyl-CoA was used to assess CPT-I activity. These modifications were made to obtain maximal reaction rates of CPT-I and to account for the effect of Nagarse on malonyl-CoA inhibition (39). The assay measures the total overt CPT activity (preincubation of mitochondria without etomoxiryl-CoA) and the etomoxiryl-CoA-insensitive CPT or overt CPT-II activity (preincubation of mitochondria with etomoxiryl-CoA). The difference between the total overt and etomoxiryl-CoA-insensitive activity represents the etomoxiryl-CoA-sensitive activity or CPT-I.

CPT-II activity was determined in both the forward (palmitoyl-L-[methyl-14C]carnitine formation from palmitoyl-CoA and L-[methyl-14C]carnitine) and reverse (CoASH-dependent release of L-[methyl-14C]carnitine from palmitoyl-L-[methyl-14C]carnitine) directions without any modification of the original procedure (24). Citrate synthase (CS) activity was determined as referenced earlier (24); succinate dehydrogenase (SDH) (23) and lactate dehydrogenase (3) activities were measured as referenced. All enzyme activities were determined at 37°C and are expressed as milliunits (nmol/min) or units (µmol/min) per mitochondrial protein or gram skeletal muscle wet weight (g wet wt). For total carnitine determination, the muscle homogenates were alkali treated to hydrolyze the acylcarnitines, and the free carnitine was quantitated by HPLC after precolumn derivatization, as described previously (38). NCP, extracted as in Ref. 32, and mitochondrial protein were determined by the Lowry method (33).

Determination of mitochondrial density and mitochondrial inner membrane intactness. The mitochondrial density (mg mitochondrial protein/g skeletal muscle wet wt) was calculated by dividing the CS and SDH activities of skeletal muscle homogenates (U/g wet wt) by the respective specific activities in isolated mitochondria (U/mg mitochondria protein). The mitochondrial recovery, expressed as a percentage, was obtained by dividing the CS and SDH activities present in isolated mitochondria equivalent to one gram of skeletal muscle wet wt (U/g wet wt) by the respective activities in skeletal muscle homogenates (U/g wet wt).

The integrity of the mitochondrial inner membrane was determined by measuring the overt CPT-II or etomoxiryl-CoA-insensitive activity in freshly isolated mitochondria (modified forward assay) and total CPT-II activity after detergent solubilization of frozen and then thawed mitochondria (forward assay). The mitochondrial inner membrane integrity is expressed as a percentage and is calculated by the following equation
Percent intactness

<IT>=</IT>(1.0<IT>−</IT>overt CPT-II activity/total CPT-II activity)<IT>×</IT>100

Oxidative phosphorylation. Oxygen consumption was measured in duplicate with each substrate by use of a Clark-type electrode in a final volume of 0.5 ml at 30°C (51). The following substrates or substrate combinations were used (in mM): glutamate (20); pyruvate (10) plus malate (5.0); palmitoyl-CoA (0.04), carnitine (2), and malate (5.0); palmitoyl-L-carnitine (0.04) plus malate (5.0); octanoyl-CoA (0.12), L-carnitine (5.0), and malate (5.0), and octanoyl-L-carnitine (0.2) plus malate (5.0). The assay was optimized to yield maximal respiratory rates with the respective substrates and carried out within 3 h after completion of mitochondrial isolation. Respiratory control ratios (state 3 rates divided by state 4 rates) and ADP-to-oxygen (ADP/O) ratios (nmol ADP consumed per nanoatom O) were calculated as referenced (8).

SDS-PAGE immunoblotting. Frozen skeletal muscle mitochondria were solubilized in SDS-PAGE sample buffer, and aliquots containing 30 µg protein were loaded per lane. SDS-PAGE and immunoblotting were carried out as described earlier (24) with the following modifications. After electrotransfer, the polyvinylidene difluoride membranes were dried and treated as in Ref. 17, and the colorimetric detection was replaced by enhanced chemiluminescent detection (Amersham Pharmacia Biotech). The primary antibodies were diluted 1,000-fold, and horseradish peroxidase-conjugated protein A was diluted 2,000-fold. Densitometric evaluation of the blots was carried out using Scion Image, a Windows-compatible version of NIH image (www.scioncorp.com).

Statistical analysis. The results are expressed as means ± SE, and the differences were compared by t-test. A difference of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondrial content and yield. To evaluate the overall oxidative capacity of skeletal muscle of adult and elderly Fischer 344 rats, we determined the mitochondrial content. We measured the activity of two exclusively mitochondrial enzymes, CS and SDH, in skeletal muscle homogenates and in isolated mitochondria, and we used these data to calculate the mitochondrial content. As shown in Table 1, the activity of both mitochondrial marker enzymes was significantly lower in skeletal muscle homogenates of 24-mo-old compared with 6-mo-old adult animals. The average decrease for both CS and SDH activities was 31%. In contrast to CS and SDH, no age-associated decrease was found in lactate dehydrogenase activity, a cytosolic marker enzyme, and in NCP in skeletal muscle homogenates (Table 1). To ascertain the reason for the lower CS and SDH activities in skeletal muscle homogenates of elderly rats, we determined the specific activities of CS and SDH in isolated mitochondria and calculated the mitochondrial content.

                              
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Table 1.   Mitochondrial and cytoplasmic marker enzyme activities and noncollagenous protein in skeletal muscle homogenates and specific activities of mitochondrial marker enzymes for 6-mo and 24-mo rats

No significant differences were observed in the specific activities of either enzyme in isolated mitochondria (Table 1). As shown in Table 1, the calculated mitochondrial content was significantly lower in skeletal muscle of elderly rats with both mitochondrial marker enzymes (25 and 20% based on CS and SDH, respectively). Nevertheless, no difference was found in the percent yield of mitochondria from skeletal muscle of adult and elderly animals (67.9 ± 4.5 vs. 75.5 ± 2.9 by CS and 79.8 ± 6.0 vs. 85.4 ± 10.3 by SDH activity, respectively). Consistent with the lower mitochondrial content, the mitochondrial protein yield was 17.2% lower from skeletal muscle of elderly rats (Table 1).

Oxidative phosphorylation. To determine the effect of aging on the overall oxidative capacity and fuel utilization of isolated skeletal muscle mitochondria, we measured polarographically the oxygen consumption with nonlipid and lipid substrates. Because the tricarboxylic acid cycle and oxidative phosphorylation represent the final common segments in the oxidative breakdown of all substrates (fatty acids, carbohydrates, and proteins), the capacity of these segments was determined separately. Glutamate was used as substrate for the respiratory chain (including glutamate uptake and glutamate dehydrogenase), and pyruvate plus malate was used for the tricarboxylic acid cycle (including pyruvate uptake and pyruvate dehydrogenase) as well as the respiratory chain. The mitochondrial fatty acid oxidation capacity was assessed with both long-chain and medium-chain acyl substrates, which for their oxidation either require (palmitoyl-, octanoyl-CoA/carnitine) or bypass CPT-I (palmitoyl- and octanoyl-carnitine). Although activated long- and medium-chain fatty acids enter the mitochondria via the same carnitine-dependent transport system, their mitochondrial oxidation differs, inasmuch as medium-chain acyl substrates enter the beta -oxidation pathway farther downstream, thus bypassing the enzymes specific for long-chain substrates. The results of these polarographic studies are presented in Table 2. There was no difference between mitochondria isolated from skeletal muscles of adult and elderly rats in state 3 respiration rates and ADP/O ratios (Table 2) with any of the substrates tested.

                              
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Table 2.   Respiratory properties of skeletal muscle mitochondria isolated from adult (6-mo) and elderly (24-mo) Fischer 344 rats

In contrast, state 4 respiratory rates were significantly lower with all substrates except glutamate (which decreased but not significantly) in mitochondria isolated from 24-mo-old animals, resulting in higher respiratory control ratios (Table 2). Possible explanations for the decrease in state 4 respiration are a decreased leak of protons back to the matrix, less damage of the inner membrane, or a lower contamination of mitochondria by ADP-regenerating systems from ATP. To evaluate the presence of a decreased proton leak catalyzed by UCPs, specifically UCP-3, the predominant isoform in skeletal muscle, we determined the content of UCP-3 in isolated skeletal muscle mitochondria. The immunoblot shown in Fig. 1A clearly reveals a significantly decreased decoration of UCP-3 in mitochondria from elderly rats. To provide a quantitative measure, the blot was scanned and the density recorded. The data in Fig. 1B show a 68% decrease in the abundance of UCP-3 in mitochondria from 24-mo-old animals. Thus these data are consistent with the observed lower state 4 respiratory rates.


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Fig. 1.   Immunoblot analysis (A) and quantification (B) of uncoupling protein-3 (UCP-3) of skeletal muscle mitochondria isolated from adult (6 mo) and elderly (24 mo) Fischer 344 rats. Lanes 1-7 and 8-14 represent mitochondria from adult and elderly animals, respectively, and nos. above bands denote density of respective bands in arbitrary units. Each lane (A) represents mitochondria (30 µg protein/lane) isolated from a separate animal. Estimation of UCP-3 abundance (B) was carried out using blot intensities (A). Nos. are means ± SE obtained with 7 mitochondrial preparations for each age group and are expressed as arbitrary units (B). #P < 0.05, significantly decreased UCP-3 abundance in mitochondria from elderly animals.

To assess a potential effect of aging on the adenine nucleotide translocase, we also determined the ADP-stimulated maximal respiratory rates (Fig. 2). As with state 3 respiration, no age-related differences were observed in maximal respiration with any of the substrates tested. Consistent with the rate-limiting role of CPT-I in overall mitochondrial fatty acid oxidation, lipid substrates, which require CPT-I for their metabolism, tended to be lower with long-chain acyl-CoA (palmitoyl-CoA) and were significantly lower with medium-chain acyl-CoA (octanoyl-CoA) compared with their respective carnitine esters, which bypass CPT-I (Fig. 2, B and C).


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Fig. 2.   ADP-stimulated maximal respiration of mitochondria isolated from skeletal muscle of adult (6-mo) and elderly (24-mo) Fischer 344 rats with nonlipid substrates (glutamate, pyruvate; A) and long- (B) and medium-chain (C) acyl-CoA and acylcarnitine substrates, respectively. Data are expressed as nanoatom O · min-1 · mg mitochondrial protein-1 and represent means ± SE of 8 and 7 animals for the 6- and 24-mo age groups, respectively. Symbols denote significantly higher respiration (P < 0.05) with octanoyl-L-carnitine (C) vs. octanoyl-CoA in both adult (#, open bars) and elderly groups (&, closed bars).

CPT activities and tissue carnitine content. CPT-I activity was determined at saturating palmitoyl-CoA concentrations (50 µM palmitoyl-CoA/0.1% BSA) in the presence of a saturating L-[14CH3]carnitine (5.0 mM) concentration in freshly isolated mitochondria and is defined as the activity inhibited by etomoxiryl-CoA. The results presented in Fig. 3A show that both the total overt CPT and etomoxiryl-CoA-suppressible or CPT-I activity are identical in skeletal muscle mitochondria from both age groups and thus are consistent with the data obtained in the oxidative phosphorylation studies that use palmitoyl-CoA plus carnitine as substrates (Table 2 and Fig. 2, B and C). Furthermore, 96 and 95% of the overt activity was inhibited by etomoxiryl-CoA in skeletal muscle mitochondria from both adult and elderly rats (e.g., little exposure of the inner membrane enzyme, CPT-II).


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Fig. 3.   Carnitine O-palmitoyltransferase (CPT)-I (A) and CPT-II (B) activities of mitochondria isolated from skeletal muscle of adult (6-mo) and elderly (24-mo) Fischer 344 rats and skeletal muscle total carnitine content (C). CPT-I and CPT-II activities were determined as described in MATERIALS AND METHODS. CPT-I activity is defined as the overt activity inhibited by etomoxiryl-CoA, a selective and irreversible inhibitor of the enzyme. In A, symbols below columns: total overt CPT (T), etomoxiryl-CoA-insensitive or overt CPT-II (II), and etomoxiryl-CoA-sensitive or CPT-I (I) activity, respectively. Values are means ± SE of 8 and 7 animals for 6- and 24-mo age groups, respectively. #P < 0.05, significantly decreased carnitine content in skeletal muscle of elderly animals.

CPT-II activity was determined on frozen and then thawed mitochondria both in the forward (palmitoylcarnitine formation) and reverse (palmitoyl-CoA formation) directions (24) under conditions providing maximal reaction rates (Fig. 3B). There was no significant age-associated difference in CPT-II activity measured in either direction. Additionally, no difference was observed in the inner membrane integrity between skeletal muscle mitochondria from adult (96% intact) and elderly (97% intact) animals as calculated from the overt (etomoxiryl-CoA-insensitive, Fig. 3A) and total CPT-II activity (Fig. 3B, forward reaction).

The skeletal muscle isoform of CPT-I has a relatively high Michaelis-Menten constant (Km) for carnitine (36), which is an obligatory cofactor of mitochondrial fatty acid oxidation. Therefore, changes in tissue free carnitine concentrations also could affect mitochondrial long-chain fatty acid oxidation. To assess this possibility, we determined the total carnitine content in skeletal muscle of adult and elderly rats. There was a moderate and significant (21%, P < 0.05) decrease in skeletal muscle total carnitine content with aging (Fig. 3C).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we observed a significantly lower content of mitochondria in skeletal muscle of elderly vs. adult Fischer 344 rats. The recovery of mitochondria through the isolation procedure from skeletal muscle of elderly and adult rats is similar; (based on CS and SDH activities, the average recovery is 80.5 and 73.9%, respectively). Thus the yield of mitochondrial protein per gram wet weight of skeletal muscle is less in elderly compared with the adult animals, consistent with the lower mitochondrial content. Lower skeletal muscle mitochondrial yield from elderly rats also has been reported by Beyer et al. (4). These authors reported a 35% decrease in mitochondrial protein yield from quadriceps femoris of elderly Sprague-Dawley rats. In contrast, no significant age-related decrease in combined mitochondrial yield (subsarcolemmal plus interfibrillar mitochondria) was found in gastrocnemius-plantaris muscle of 24-mo-old Sprague-Dawley rats by Farrar et al. (16).

The age-related loss of mitochondria documented in the present study is not a consequence of a general decrease in protein content, because neither NCP nor lactate dehydrogenase activity, a cytosolic marker enzyme, was affected. Although skeletal muscles from senescent animals have fewer mitochondria, the recovery of these organelles from muscles of adult and elderly rats was virtually identical, thus arguing against an increased fragility of mitochondria from senescent animals. This conclusion is further supported by the high membrane integrity of the skeletal muscle mitochondria isolated from both age groups, with <4% of their inner membrane disrupted.

We have isolated the mitochondria from skeletal muscle of both hindlimbs, which represents up to 50% of the total muscle mass of a rat and comprises a wide range of fiber types. Thus the decrease in mitochondrial density observed in this study represents an "average" decline. Because the effect of aging on skeletal muscles is muscle and fiber type specific (21), it is reasonable to assume that the mitochondrial density of some muscles is more affected than that of others. Furthermore, skeletal muscle mitochondria exist as two distinct populations (subsarcolemmal mitochondria and interfibrillar mitochondria) (41) and are known to respond differently to different physiological and pathological stimuli (15, 28, 53). Whether aging also affects subsarcolemmal and interfibrillar mitochondria to a different extent in skeletal muscle remains to be determined. The limited data on the effect of aging on the content of subsarcolemmal and interfibrillar mitochondria would support this notion (16).

It has been suggested that decreased physical activity with aging might cause, in part, some of the age-related changes in hindlimb muscles. Both spontaneous wheel running (22) and spontaneous in-cage activity (58) decline with age. Studies on the effect of exercise training on markers of skeletal muscle respiratory capacity, measured on whole tissue homogenates, demonstrate that the differences in muscle respiratory capacity between young and old animals are greatly attenuated or even eliminated (4, 7, 16, 43). From these data, it has been inferred that the decreased age-related aerobic skeletal muscle capacity is a result of reduced physical activity. However, other studies demonstrated that skeletal muscles of elderly rats have an attenuated response to exercise, indicating that the reduced physical activity is not the only contributing factor to the age-related loss of skeletal muscle function (35, 56). Consistent with the latter interpretation is the dramatic age-associated decrease in UCP-3 abundance (despite the lack of changes in specific activities of mitochondrial enzymes) observed in the present study and in the study by Barazzoni and Nair (1)1. In further support of this notion are data on UCP-3 mRNA expression in skeletal muscle of Wistar rats after a 5-wk hindlimb unloading, a model of long-term reduced physical activity (12). These authors found that, whatever the duration of mechanical unloading, UCP-3 was upregulated in soleus, with no change in expression in extensor digitorum longus. Thus these data suggest that age-associated changes in skeletal muscle mitochondria do occur and can be dissociated from the level of physical activity.

To determine whether aging has a selective impact on skeletal muscle fuel utilization, isolated mitochondria were subjected to functional studies by measuring their respiratory properties by use of nonlipid substrates (glutamate and pyruvate plus malate) and CPT-I-dependent (palmitoyl-CoA) and CPT-I-independent (palmitoyl-L-carnitine) lipid substrates. In addition to long-chain lipid substrates, we also used the respective octanoyl derivatives (medium-chain lipid substrates) to test whether there is an age-related decrease in the mitochondrial beta -oxidation farther downstream from long-chain acyl-CoA dehydrogenase and the trifunctional enzyme protein. In contrast to the significant age-related decrease in mitochondrial content, with the exception of state 4 respiration, the respiratory properties of mitochondria isolated from 24-mo-old animals were indistinguishable from those isolated from 6-mo-old adult animals with all substrates tested in this study. There was no age-associated decrease in state 3 respiration, in maximal ADP-stimulated respiration, and in ADP/O ratios; hence the data argue against age-related deterioration in the potential of skeletal muscle mitochondria to oxidize different substrates and to synthesize ATP.

However, state 4 respiration was significantly decreased in skeletal muscle mitochondria from elderly rats, thus resulting in elevated respiratory control ratios. State 4 rates are governed by the proton leak across the inner membrane mediated by UCPs, the intactness of the inner membrane, and the presence of ADP-generating systems (ATPases). An increased inner membrane intactness seems unlikely, because the inner membrane integrity is virtually identical in skeletal muscle mitochondria from adult and elderly rats (e.g., 96 and 97% intact, respectively). On the basis of the UCP-3 knockout mouse with decreased state 4 rates but otherwise normal state 3 respiration (55), we focused on the decrease in UCP-3 (the predominant isoform in skeletal muscle). Consistent with the lower state 4 respiratory rates of skeletal muscle mitochondria from elderly rats, there is a 68% decrease in UCP-3 protein abundance compared with mitochondria from adult rats. If we consider the proposed role(s) of UCP-3 to influence metabolic rate (10, 14, 31) and lipid metabolism (14 and references therein), from a decreased UCP-3 expression in skeletal muscle we would have anticipated an excessive weight gain with age. However, Fischer 344 rats gain weight only moderately with age compared with other strains (i.e., Sprague-Dawley, Wistar, Long Evans). Furthermore, a direct role of UCP-3 in mitochondrial fatty acid oxidation, the major energy-producing pathway in muscle, also seems unlikely, because no age-related change in beta -oxidation was noted (see further discussion). Our data on state 4 respiration and UCP-3 protein abundance are rather in line with recent findings reported by Vidal-Puig et al. (55). Skeletal muscle mitochondria from UCP-3 knockout mice display respiratory properties similar to those isolated from skeletal muscles of elderly rats in the present study, i.e., unchanged state 3 and decreased state 4 respiration and thus higher respiratory control rates compared with the wild-type mice (55). Although the biological significance of the latter finding is unclear, an increased coupling could result in an increased proton electrochemical gradient and, consequently, in increased production of reactive oxygen species. In keeping with such a situation are findings of increased free radical production in mitochondria isolated from skeletal muscles of UCP-3 knockout mice as opposed to those isolated from wild-type mice (55), as well as of skeletal muscle mitochondria isolated from elderly vs. adult Fischer 344 rats (2). Further support for a role of UCP-3 in regulation of mitochondrial proton leak has been provided by Gong et al. (18), who demonstrated an increased protonmotive force in skeletal muscle mitochondria from UCP-3 knockout mice. Although in light of the mitochondrial free radical theory of aging it is tempting to speculate that there is a cause-effect relationship between increased free radical production and decreased mitochondrial density with advanced age, the unimpaired exercise tolerance of UCP-3 knockout mice (55) does not support this hypothesis.

Although there was no age-associated change in the respiratory properties of isolated mitochondria with substrates, lipid substrates that bypass CPT-I and enter the mitochondria via the acylcarnitine:carnitine translocase and CPT-II (palmitoyl-L-carnitine, octanoyl-L-carnitine) showed higher state 3 and ADP-stimulated (2 mM) maximal respiratory rates than those that for their mitochondrial uptake also required CPT-I (palmitoyl-CoA, octanoyl-CoA). Significantly higher state 3 respiratory rates with palmitoylcarnitine as opposed to palmitoyl-CoA plus carnitine were also reported by Chen et al. (9). These data are consistent with the notion that CPT-I is rate determining in the mitochondrial oxidation of activated fatty acids.

We also determined the activities of CPT-I and CPT-II in isolated mitochondria as well as tissue carnitine content. The activities of both enzymes were measured under conditions that provide maximal reaction rates. Consistent with the polarographic studies with CPT-I substrates, no age-related changes were found in maximal enzyme activity. These data are in contrast to the age-related decrease in CPT-I activity described in isolated rat and mice heart mitochondria (37, 40). Also, no differences were found in CPT-II activity, whether measured in the forward or reverse direction, again consistent with the polarographic studies with substrates requiring the acylcarnitine:carnitine translocase and CPT-II activities but not CPT-I.

Skeletal muscle carnitine contents are relatively high and thus are not thought to limit fatty acid oxidation under normal physiological conditions. However, the skeletal muscle isoform of CPT-I has a relatively low affinity for carnitine (36), with a Km value (~500 µM) within the reported range of free carnitine concentrations in skeletal muscle (27, 46). Extrapolating these values to in vivo conditions, CPT-I is operating under nonsaturating conditions with respect to carnitine, and, hence, changes in free carnitine concentrations could have an impact on the rate of mitochondrial fatty acid oxidation. Therefore, we also determined the skeletal muscle carnitine content as a function of age. Because it is unlikely that the in vivo acylation status of carnitine is preserved after Nembutal anesthesia and muscle dissection (27), only total carnitine was determined. Nevertheless, changes in total carnitine concentration should be reflected by appropriate directional changes in free carnitine. In line with data published by others (7, 11, 20), we found a moderate but significant decrease in total carnitine in skeletal muscles of old animals (from 874.7 ± 67.0 to 683.2 ± 28.7 nmol/g wet wt). In rat heart, ~90% of the total carnitine is in the cytoplasm (26). If we assume a similar subcellular distribution in rat skeletal muscle and take into account that ~20-30% of total carnitine is present as acetylcarnitine in resting skeletal muscle (27), even relatively small changes in total carnitine content, as observed in this study as well as in Refs. 7, 11, and 20, could impair mitochondrial fatty acid oxidation. In support of this hypothesis are recent in vivo human studies demonstrating parallel changes in skeletal muscle fatty acid oxidation and free carnitine concentrations during exercise (54).

In conclusion, we have shown that skeletal muscle mitochondrial density decreases significantly with age. In vivo, the decreased aerobic capacity of skeletal muscle with advanced age is a consequence of the decreased mitochondrial density and in addition may be further compounded by the well documented loss of skeletal muscle mass. The decrease in UCP-3 in skeletal muscle mitochondria leads to a decrease in state 4 respiration, with no effect on state 3 respiratory rate. The physiological consequence of this observation deserves further study. Our data on isolated skeletal muscle mitochondria do not support the notion that the age-related decrease in skeletal muscle function is a consequence of impaired mitochondrial oxidative capacity. Although we were unable to detect any age-related change in CPT-I activity, the key enzyme in overall mitochondrial fatty acid oxidation, the observed decrease in total carnitine with advanced age could impair skeletal muscle fatty acid utilization.


    ACKNOWLEDGEMENTS

We thank Drs. Edward Lesnefsky and Bernard Tandler for reviewing the manuscript.


    FOOTNOTES

This research was supported by a pilot project grant from Case Western Reserve University Pepper Older Americans Independence Center; National Institute on Aging Grants P60 AG-10418, POI AG-15885, and 1-R01 AG-12447 (awarded to E. J. Lesnefsky); and the Medical Research Service of the Department of Veterans Affairs.

Part of this work was presented at the Experimental Biology 2000 meeting in San Diego, CA, and was published in abstract form in FASEB J 14: A300, 2000.

Address for reprint requests and other correspondence: J. Kerner, Louis Stokes Cleveland VA Medical Center, Medical Research Center (151W), 10701 East Boulevard, Cleveland, OH 44106 (E-mail: jxk81{at}po.cwru.edu).

1  While this manuscript was under review, R. Barazzoni and K. S. Nair reported an age-related decrease in UCP-3 mRNA levels in rat gastrocnemius muscle that was more pronounced in the less oxidative white (medial head) than in the more oxidative red fibers (lateral head). At the protein level, there was a 30% reduction in mixed gastrocnemius muscle. In contrast to UCP-3, both transcript and protein levels of UCP-2 were upregulated.

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.

Received 5 March 2001; accepted in final form 1 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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