1Department of Physiology, Brody School of Medicine, and 2Department of Exercise and Sport Science, College of Health and Human Performance, East Carolina University, Greenville; and 3Department of Medicine, 4Department of Pharmacology and Cancer Biology, and 5Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, North Carolina
Submitted 9 August 2004 ; accepted in final form 4 January 2005
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ABSTRACT |
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endurance exercise training; CPT-1; fiber type; rat; mitochondrial subpopulations
Studies on mitochondrial function in skeletal or cardiac muscle present the added dimension of two distinct mitochondrial subpopulations that might exist as a continuous reticulum (25, 30) but differ according to their subcellular localization, morphology and biochemical properties (8, 33, 42). Subsarcolemmal (SS) mitochondria are located just underneath the sarcolemma and have a large, lamellar shape. In contrast, the intermyofibrillar (IMF) mitochondria are smaller, more compact, and located between the contractile filaments. Several studies have shown that SS and IMF mitochondria possess inherent differences in their capacity to adapt to changes in contractile activity (5, 21, 31). Compared with IMF mitochondria, the SS mitochondria display greater exercise-induced increases in volume, state III respiration, and enzyme activities (5, 31). The SS mitochondria also show more pronounced decrements in response to muscle disuse (31).
The present study sought to determine whether these two subpopulations play discrete roles in mediating training-induced increases in skeletal muscle fatty acid oxidation, and/or in conferring the distinct metabolic properties of red (type I) compared with white (type II) fiber types. In a previous report we provided evidence of fiber type-dependent differences in the kinetic properties of the muscle isoform of carnitine palmitoyltransferase-1 (CPT1
) (29), the enzyme that controls mitochondrial uptake of fatty acid and functions as the rate-determining step in
-oxidation. CPT1
localizes to the outer mitochondrial membrane and is largely regulated by cytosolic concentrations of its biological inhibitor malonyl-CoA. However, we identified a malonyl-CoA-insensitive CPT1
fraction that is predominately active in mitochondria from red skeletal muscle (29). In the present study, we investigated whether this malonyl-CoA-resistant CPT1
subfraction might be differentially active in SS compared with IMF mitochondria. Furthermore, because endurance training is known to increase mitochondrial oxidative capacity (i.e., metabolic shift toward enhanced lipid utilization), we also tested whether this expansion in capacity is generalized or specific to a given mitochondrial subpopulation. Our results indicate that the oxidative properties of muscle mitochondria depend on fiber type and are enhanced by endurance training. Moreover, we show that SS and IMF mitochondria play distinct metabolic roles in mediating both fiber type-specific and exercise-induced regulation of CPT1
and fatty acid oxidation. These findings support the emergent view that skeletal muscle possesses distinct mitochondrial subpopulations that are inherently unique in different muscle fiber types and that display distinct adaptations in response to energy stress.
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MATERIALS AND METHODS |
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Exercise protocol. Animals weighing 200225 g were randomly assigned to either a training (n = 7) or control (n = 7) group. Earlier, Molé and Holloszy (39) demonstrated the absence of a difference in palmitate oxidation between ad libitum-fed controls and control pairs fed to match body weights of the trained group. Therefore, both trained and sedentary animals were allowed ad libitum access to food and water. The rats in the training group were habituated to the treadmill over a 2-wk period to final conditions of 35 m/min and 8% grade. Rats were run 1 h/day, 5 days/wk, for a period of 1012 wk. Similar training protocols have been shown to produce increases in the capacity of skeletal muscle to oxidize fatty acids (2, 40). Animals were euthanized 48 h after their last training bout.
Muscle sample preparation. Animals were administered a mixture containing 90 mg/ml ketamine and 10 mg/ml xylazine at 0.1 ml·100 g body wt1 and muscles were quickly excised, cleaned, rinsed, blotted, weighed, and placed into ice-cold modified Chappel-Perry buffer (solution 1) consisting of (in mM) 100 KCl, 40 Tris·HCl, 10 Tris-Base, 5 MgCl2, 1 EDTA, and 1 ATP, pH 7.5.
Mitochondrial preparations.
Mitochondrial suspensions were prepared according to the methods of Bizeau et al. (5) and Palmer et al. (42) with modifications for the preparation of IMF mitochondria. To prepare the SS mitochondria, gastrocnemius samples were placed in fresh solution 1 (see above) so that they were twofold diluted (wt/vol), minced, suspended 10-fold (wt/vol) in solution 1, and homogenized at 40% power (9,500 rpm) in an Ultra Turrax homogenizer (model T-25; IKA, Wilmington, NC) for 10 s. The homogenate was centrifuged at 800 g for 10 min at 4°C. The supernatant was filtered through double-layered gauze and centrifuged at 9,000 g to pellet the SS mitochondria. The pellet was gently resuspended in a buffer (solution 2) consisting of (in mM) 100 KCl, 10 Tris·HCl, 10 Tris-Base, 1 MgSO4, 0.1 EDTA, 0.02 ATP, and 1.5% BSA, pH 7.4, and centrifuged at 9,000 g. The pellet was washed and gently resuspended in a buffer (solution 3) with the same components as solution 2, except without BSA and centrifuged for 10 min at 5,000 g. The final pellet was resuspended in 0.5 to 1.3 ml of suspension buffer consisting of (in mM) 250 sucrose, 10 Tris·HCl, 1 EDTA, and 2 ATP, pH 7.4.
IMF mitochondria were initially prepared by two different methods: 1) according to the methods of Bizeau et al. (5) using the bacterial protease Nagarse and 2) by a mechanical method modified from the one described by Brooks et al. (6). Mitochondrial fatty acid oxidation rates were not affected by the isolation methods. However, we did find that malonyl-CoA was completely ineffective at inhibiting fatty acid oxidation in IMF mitochondria that were prepared using Nagarse, whereas those prepared mechanically retained this inhibition. Subsequent studies were therefore performed with the use of the mechanical method. In brief, the 800 g-centrifuged pellet described above was resuspended 10-fold in solution 1. The homogenate was transferred to a 30-ml Potter-Elvehjem glass homogenization vessel, homogenized (10 passes across 30 s at 1,000 rpm), and then centrifuged at 800 g for 10 min at 4°C. The supernatant was filtered through double-layer gauze, and mitochondria were pelleted, washed, and resuspended as described for SS mitochondria.
Oxidation studies. Oxidation studies on muscle mitochondria were performed according to methods originally described by Veerkamp et al. (51) and modified by Kim et al. (28). In brief, 40 µl of the final mitochondrial suspensions were incubated in the presence of 160 µl of an oxidation buffer containing (in mM) 0.150 palmitate ([1-14C]palmitate at 0.5 µCi/ml1; PerkinElmer Life Sciences), 100 sucrose, 10 Tris·HCl, 10 KH2PO4, 100 KCl, 1 MgCl2, 1 L-carnitine, 0.1 malate, 2 ATP, 0.05 CoA, 1 DTT, and 0.3% BSA and either 0 or 0.01100 µM malonyl-CoA (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C. Specific activity was maintained between 7,000 and 8,000 dpm·nmol palmitate1 for all experiments. The reaction was terminated by the addition of 100 µl of 70% perchloric acid. 14CO2 was trapped in 200 µl of 1 N NaOH. Radioactive 14CO2 was assessed by counting 150 µl from NaOH traps by liquid scintillation counting in 4 ml of Uniscint BD (National Diagnostics, Atlanta, GA).
For these experiments, palmitate oxidation and the subsequent inhibition by malonyl-CoA was used as a surrogate for CPT1 activity. This strategy was based on the observation that CPT activity can be measured in both microsomal and mitochondrial fractions as well as in mitochondrial fragments, in which malonyl-CoA insensitive CPT2 might be exposed (36). Traditionally, CPT1 specific activity is calculated by subtracting the residual activity (presumably attributed to CPT2) that is measured in the presence of a maximally inhibiting dose of malonyl-CoA. In contrast, we quantified 14C-labeled CO2, produced only from intact mitochondria, as an index of palmitate oxidation.
Enzyme activity assays.
Muscle enzyme activities were determined from 10 µl (10 µg protein) of the mitochondrial suspensions or whole tissue homogenates. Protein concentrations were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL). CPT1
activity was determined using modifications of the methods of McGarry et al. (37) at 37°C for 6 min, a time at which we observed that the formation of [3H]palmitoyl-carnitine remained linear. [3H]palmitoyl-carnitine formed was extracted with 350 µl of water-saturated butanol and quantified by liquid-scintillation counting. Citrate synthase (CS) activity was assessed in a 10-fold diluted mitochondrial suspension using the spectrophotometric method of Srere (49).
Analyses. All data are reported as means ± SE. Differences between mitochondrial subpopulations due to training were analyzed using one-way ANOVA. Fiber type and subpopulation differences were analyzed using two-way ANOVA. When significant differences were observed, comparisons between mean values were made using a Student-Newman-Keuls post hoc test. Significance levels were set a priori at P < 0.05 for all comparisons. In the malonyl-CoA inhibition studies, IC50 values and Hill coefficients were calculated using Prism software (GraphPad, San Diego, CA) with parameters set for best-fit nonlinear regression equations and variable slope constraints. Differences between IC50 values and Hill coefficients were calculated using unpaired t-tests and Welch's corrections if variances were unequal. All other statistical analyses were also performed using GraphPad Prism.
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RESULTS |
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Mitochondrial yields using mechanical methodologies were 0.69 ± 0.04 vs. 0.91 ± 0.05 mg protein·g tissue1 for deep red gastrocnemius muscle SS and IMF mitochondria, respectively, and 0.46 ± 0.05 vs. 0.44 ± 0.03 mg protein·g tissue1 for superficial white gastrocnemius muscle SS and IMF mitochondria, respectively. Rates of palmitate oxidation were similar between IMF and SS mitochondria when normalized to mitochondrial protein (Fig. 1). Remarkably, however, palmitate oxidation was 8.9-fold higher (P < 0.05) in SS mitochondria and 5.3-fold higher (P < 0.05) in IMF mitochondria that were isolated from red compared with white gastrocnemius muscle, respectively, thus demonstrating striking fiber type-dependent differences in the fatty acid oxidative capacity of muscle mitochondria. In addition, in SS mitochondria from red gastrocnemius, 10 µM malonyl-CoA inhibited palmitate oxidation 89%, whereas the same dose of malonyl-CoA inhibited IMF mitochondria from red gastrocnemius by only 60% (P < 0.01; Fig. 2A). Conversely, when mitochondria were prepared from white gastrocnemius, malonyl-CoA inhibitable palmitate oxidation was similar in SS and IMF mitochondria (Fig. 2B). These data show that both SS and IMF mitochondria from type I muscle fibers display a markedly higher capacity to oxidize fatty acid compared with the respective subpopulations in type II fibers, and that malonyl-CoA-mediated inhibition of CPT1 depends on properties that are affected by both fiber type and mitochondrial subfraction.
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DISCUSSION |
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In agreement with previous reports, we found that both CPT1 (4) and CS activities (8) were similar in IMF compared with SS mitochondria from rat gastrocnemius muscle. Our finding that training-induced increases in some oxidative enzyme activities were more robust when measured in whole homogenates than in isolated mitochondria is also consistent with previous reports (19, 40) and suggests that some changes in oxidative phosphorylation potential observed at the whole muscle level are in fact due largely to mitochondrial expansion. To the contrary, data from isolated mitochondria indicate that other adaptations, such as increased lipid oxidation, appear to reflect both an increase in mitochondrial number and a change in mitochondrial function.
Consistent with other studies reporting that SS mitochondria appear to be more responsive than the IMF population to endurance training (5, 31), we found that endurance exercise training increased palmitate oxidation rates 100% in SS but only 46% in IMF mitochondria from the gastrocnemius muscle. Thus the paradigm observed in our exercise studies, in which training markedly enhanced the fatty acid oxidation rates of mixed muscle, resembled that in the fiber type model, in which fatty acid oxidation capacity was 9-fold higher in SS and
5-fold higher in IMF mitochondria from red compared with white skeletal muscles. These observations are consistent with the notion that endurance training causes a metabolic remodeling of white and intermediate muscle fibers, which in turn produces a phenotype that more closely resembles that of red muscle.
To our knowledge, this is the first study to specifically examine the lipid oxidative capacity of mitochondrial subpopulations from red, white, and mixed skeletal muscle. When normalized for mitochondrial protein, the oxidative capacities between subpopulations appeared similar in extreme red and similar or slightly higher in extreme white muscle IMF mitochondria. However, this was not the case in whole gastrocnemius muscle, in which the oxidative capacity of IMF mitochondria was higher than it was in SS mitochondria. The whole gastrocnemius consists of a more heterogeneous population of fiber types than either the red or white portions of the muscle. Delp and Duan (12) reported that the fiber type composition of extreme red gastrocnemius is 51, 35, 13, and 1% (types I, IIa, IId/x, and IIb), respectively, and that that of extreme white gastrocnemius is 0, 0, 8, and 92%. The fiber type distribution of whole gastrocnemius (3, 6, 34, and 57%) is closer to that of extreme white muscle but exhibits a higher proportion of type IId/x fibers than both extreme white and extreme red muscle. Interestingly, CS activities (36.2, 25.7, and 8.1 µmol·min1·g tissue1 for extreme red, whole, and extreme white gastrocnemius, respectively) suggest that whole gastrocnemius behaves metabolically more like red muscle, despite its fiber type distribution. Thus our results are in agreement with previous studies (41) showing that fiber type composition and metabolic activity are not perfectly correlated, and they suggest that the type IId/x fibers may contain a population of highly oxidative IMF mitochondria.
Our results showed that CPT1 activity was similar between the SS and IMF mitochondria subfractions and between mitochondria from control compared with trained muscles. However, under physiological conditions, this enzyme is largely controlled by cytosolic concentrations of malonyl-CoA. We therefore sought to determine whether the CPT1
enzymes residing in these two mitochondrial subfractions might be differentially resistant to malonyl-CoA. In a previous report (29), we demonstrated that red muscle exhibited a relative malonyl-CoA insensitivity compared with white skeletal muscle. In the present study, we show that the malonyl-CoA-resistant property of red muscle is most predominant in the IMF subfraction (Fig. 2A). Moreover, in the IMF fraction, exercise training altered malonyl-CoA inhibition of palmitate oxidation in a manner that lowered the Hill coefficient. These data suggest that fatty acid oxidation in IMF mitochondria of trained muscle is less responsive to changes in malonyl-CoA content, possibly because of adaptations that impart negative cooperativity (e.g., when binding of a substrate or inhibitor decreases an enzyme's binding affinity for subsequent molecules of its substrate or inhibitor).
In the absence of malonyl-CoA, CPT1 dependence on palmitoyl-CoA has been shown to follow a hyperbolic relationship. The addition of malonyl-CoA produces a more sigmoid function, which is thought to reflect a more finely regulated system. Our understanding of CPT1 kinetics is further complicated by evidence suggesting that malonyl-CoA can act as both a competitive (38) and an allosteric (11) inhibitor of the enzyme. Finally, because CPT1 has binding sites for carnitine, malonyl-CoA, and acyl-CoA, altered binding of any of these molecules could have contributed to the apparent negative cooperativity observed in IMF mitochondria from trained muscle. This finding might explain how exercise training provokes a disconnect between muscle malonyl-CoA levels and -oxidative flux (22). One intriguing possibility is that exercise-mediated alterations in malonyl-CoA inhibition kinetics might shift the regulation of
-oxidation away from cytosolic levels of malonyl-CoA and toward substrate delivery of acyl-CoAs, which is enhanced after exercise training. A complete understanding of the molecular mechanisms that regulate muscle CPT1 activity and sensitivity to malonyl-CoA is still unfolding. Interestingly, a recent report showed that phosphorylation of CPT1
(the liver isoform) by a cAMP-dependent kinase alters both enzyme activity and malonyl-CoA inhibition kinetics (27). The relevance of this mechanism in skeletal muscle is yet unknown.
Our present study also shows that exercise training had no effect on the IC50 value of malonyl-CoA in either mitochondrial subpopulation. This finding is in general agreement with one study showing that training had no effect on malonyl-CoA sensitivity (15), but contrasts with another report indicating that training increased CPT1 sensitivity to malonyl-CoA in human muscle mitochondria (50). In these previous studies, CPT1 activity assays were performed on total mitochondria (15) or in only one subpopulation with limited doses of malonyl-CoA (50). Furthermore, Hill coefficients, and thus possible changes in cooperativity, were not determined. In comparison, we performed rigorous biochemical analyses that evaluated malonyl-CoA-mediated regulation of a multistep physiological outcome (e.g., complete fatty acid oxidation to CO2). Importantly, this methodology eliminated inadvertent measurement of CPT2, which resides on the inner mitochondrial membrane and is insensitive to malonyl-CoA (37). In previous reports (37), residual CPT activity in muscle mitochondria challenged with high concentrations of malonyl-CoA was considered to be an artifactual activity resulting from exposure of CPT2 during mitochondrial processing. However, exposed CPT2 cannot explain our results, because we evaluated malonyl-CoA-mediated inhibition of palmitate oxidation. We consider it unlikely that mitochondrial damage could have occurred in a manner that differed systematically between mitochondrial preparations and in such a way as to permit CPT1
-independent palmitoyl-CoA entry into the matrix without coincident disruption of both the
-oxidative and TCA pathways. In addition, polarography experiments that used succinate as a substrate showed similar respiratory coupling ratios (RCR) between SS mitochondria (RCR = 6.4) and IMF mitochondria (RCR = 6.6) (data not shown), and we demonstrated that palmitate oxidation was fully dependent upon the presence of both carnitine and CoA (Table 1). Together, these data provide strong evidence that our isolation protocols maintained an intact outer membrane and malonyl-CoA responsiveness in both SS mitochondria and IMF mitochondria.
A fundamental question that arises from this study is why mitochondria from exercise trained muscle, and/or muscles rich in type I fibers, exhibit an inherent enhanced capacity to oxidize lipid substrate. Perhaps these functional distinctions relate to properties of the mitochondrial membrane environment, potentially imparted by fiber type- or subpopulation-specific differences in membrane lipid composition and/or import of proteins such as UCP3 and CD36 (7, 23, 32). Another intriguing aspect of skeletal muscle mitochondrial biology centers on speculation that these two subfractions perform specific bioenergetic roles in providing ATP for compartmentalized energy requirements. For example, SS mitochondria might exist near the sarcolemma to provide ATP for processes related to membrane function and transport, whereas IMF mitochondria might support contractile activity (34). Evidence from the current study as well as previous work indicates that the two subpopulations are differentially responsive to common stimuli, thus implying the existence of distinct, compartmentalized signaling pathways that link biogenesis of specific mitochondrial subpopulations to localized ATP demands. Notably, SS mitochondria were specifically expanded in a transgenic mouse model that is characterized by muscle fiber type conversions resembling those that occur in response to exercise training (52). In these animals, muscle-specific expression of a constitutively active form of calcium/calmodulin-dependent kinase IV results in the robust induction of peroxisome proliferator-activated receptor- co-activator-1
(PGC1
), a master transcriptional co-activator that promotes mitochondrial biogenesis. PGC1
levels have also been shown to be increased in more oxidative muscle fibers. Both calcium-mediated signals and PGC1
have been implicated in regulating metabolic adaptations and fiber type conversion in muscle due to increased contractile activity. It is therefore tempting to speculate that the functional changes in SS mitochondria that we observed in response to training might be specifically mediated by exercise-induced activation of the calmodulin-dependent kinase/PGC1
pathway.
The broader and perhaps more therapeutically relevant implications of this study relate to the emerging link between muscle lipid homeostasis, mitochondrial function, and whole body metabolic fitness. Human studies have shown that metabolic diseases such as obesity and diabetes are associated with a low percentage of type I (red) muscle fibers (16). Conversely, compared with their wild-type counterparts, mice that are genetically engineered to have a high number of type I fibers are more insulin sensitive and less susceptible to diet-induced obesity (46). In rodents (44, 47) and humans (3), both age-related and diet-induced insulin resistance have been associated with elevated or dysregulated muscle levels of malonyl-CoA. Genetic manipulations that lower muscle malonyl-CoA content increase lipid catabolism and protect against obesity (1). Together with our data, these observations suggest that the insulin-sensitizing effects of regular physical activity might be attributable, at least in part, to an increase in type I fibers and the corresponding expansion of a mitochondrial population that is less responsive to malonyl-CoA.
In addition to their principal tasks of substrate oxidation and ATP generation, mitochondria are also a primary source of reactive oxygen species (ROS) and play major roles in antioxidant defense and programmed cell death. Increased oxidative stress can lead to the production of ROS, which have been implicated in the pathogenesis and subsequent complications of aging (43). Cross-sectional studies have found that muscle lipid peroxidation (a marker of oxidative stress) is lower in endurance-trained subjects compared with either obese or insulin-resistant subjects, despite similarly high intramuscular triglyceride levels among the three groups (45). In addition, both acute (35) and chronic exercise (17) have been shown to elicit adaptations that boost antioxidant defense mechanisms, thereby conferring protection against oxidative stress and ROS-induced cellular damage. For example, exercise causes the robust induction of the muscle uncoupling proteins UCP2 and UCP3, which are thought to function in a protective manner by limiting the generation of ROS (48). Notably, one study (23) also reported that UCP3 protein content is greater in SS compared with IMF mitochondria. In the aggregate, these results support the notion that exercise-mediated changes in mitochondrial numbers and/or function might serve to protect against oxidative stress (45) and, moreover, suggest that the SS and IMF mitochondrial subpopulations may indeed play distinct roles in ROS generation and/or antioxidant defense.
In conclusion, we have demonstrated that distinct mitochondrial subpopulations display both quantitative and qualitative metabolic differences that are dependent upon muscle fiber type and training status. Our results, which support the idea that SS mitochondria and IMF mitochondria might perform distinct bioenergetic functions within the muscle, now prompt provocative new questions with respect to the potential roles of these two subpopulations in mediating exercise-induced protection against age- and disease-related metabolic dysfunction (13, 26).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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