Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels ( approximately 65% maximal O2 uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was approximately 25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 +/- 46 vs. 280 +/- 51; Sol: 390 +/- 81 vs. 368 +/- 82; RG: 252 +/- 71 vs. 278 +/- 44 nmol.min-1.mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by approximately 34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.     

Lack of REDD1 reduces whole body glucose and insulin tolerance,and impairs skeletal muscle insulin signaling

A lack of the REDD1 promotes dysregulated growth signaling, though little has been established with respect to the metabolic role of REDD1. Therefore, the goal of this study was to determine the role of REDD1 on glucose and insulin tolerance, as well as insulin stimulated growth signaling pathway activation in skeletal muscle. First, intraperitoneal (IP) injection of glucose or insulin were administered to REDD1 wildtype (WT) versus knockout (KO) mice to examine changes in blood glucose over time. Next, alterations in skeletal muscle insulin (IRS-1, Akt, ERK 1/2) and growth (4E-BP1, S6K1, REDD1) signaling intermediates were determined before and after IP insulin treatment (10 min). REDD1 KO mice were both glucose and insulin intolerant when compared to WT mice, evident by higher circulating blood glucose concentrations and a greater area under the curve following IP injections of glucose or insulin. While the REDD1 KO exhibited significant though blunted insulin-stimulated increases (p < 0.05) in Akt S473 and T308 phosphorylation versus the WT mice, acute insulin treatment has no effect (p < 0.05) on REDD1 KO skeletal muscle 4E-BP1 T37/46, S6K1 T389, IRS-1 Y1222, and ERK 1/2 T202/Y204 phosphorylation versus the WT mice. Collectively, these novel data suggest that REDD1 has a more distinct role in whole body and skeletal muscle metabolism and insulin action than previously thought.     

Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle

Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.     

Modest PGC-1alpha overexpression in muscle in vivo is sufficient to increase insulin sensitivity and palmitate oxidation in subsarcolemmal, not intermyofibrillar, mitochondria

PGC-1alpha overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1alpha overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1alpha overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1alpha overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1alpha was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5'-AMP-activated protein kinase, alpha2 subunit, and HSL. Transfection of PGC-1alpha into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1alpha protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5'-AMP-activated protein kinase, alpha2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1alpha overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1alpha overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.     

Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity

We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis.     

Respiratory activity,energy and redox status in sulphur-deficient bean plants

Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic processes. S-deficient bean plants (Phaseolus vulgaris L. cv. Złota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower dry matter production. Phenotypic effects of S deficiency appeared as depressed shoot growth, paling and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show that S deficiency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex I. Increased activities of internal NADH dehydrogenases (ND_in) may at least partially compensate for Complex I impairment. External NADH dehydrogenases (ND_ex) activities, as well as protein level and capacity of alternative oxidase (AOX), did not change in S-deficient bean plants. Total ATP concentration severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-deficient bean plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our findings indicate that flexible function of plant mitochondrial respiratory chain could be an important target during adaptations to S deficiency.     

Unsaturation of mitochondrial membrane lipids is related to palmitate oxidation in subsarcolemmal and intermyofibrillar mitochondria

Membrane lipid composition is thought to influence the function of integral membrane proteins; however, the potential for lipid composition to influence overall mitochondrial long-chain fatty acids (LCFA) oxidation is currently unknown. Therefore, the naturally occurring variability of LCFA oxidation rates within subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in muscles with varying oxidative potentials (heart → red → white) was utilized to examine this relationship. To this end, SS and IMF mitochondria were isolated and palmitate oxidation rates were compared to membrane phospholipid composition. Among tissues, rates of palmitate oxidation in mitochondria displayed a 2.5-fold range, creating the required range to determine potential relationships with membrane lipid composition. In general, the percent mole fraction of phospholipid head groups and major fatty acid subclasses were similar in all mitochondria studied. However, rates of palmitate oxidation were positively correlated with both the unsaturation index and relative abundance of cardiolipin within mitochondria (r = 0.57 and 0.49, respectively; p < 0.05). Thus, these results suggest that mitochondrial LCFA oxidation may be significantly influenced by the total unsaturation and percent mole fraction of cardiolipin of the mitochondrial membrane, whereas other indices of membrane structure (e.g., percent mole fraction of other predominant membrane phospholipids, chain length, and ratio of phosphatidylcholine to phosphatidylethanolamine) were not significantly correlated.     

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.     

Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting.

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.     

Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status, and ion transporter gene expression

Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.     

Quantitative and qualitative adaptation of skeletal muscle mitochondria to increased physical activity

Endurance capacity rely on high muscle oxidative capacity but should also involve a tighter coupling between energy production and utilization within the myocyte. The present study examined the responses of muscle oxidative capacity and the regulation of oxidative phosphorylation by phosphate acceptors in locomotor muscles of voluntary running rats (n = 8), using saponin permeabilized fibers of the deep and superficial parts of plantaris muscle (dPLA and sPLA, respectively). Non-ADP stimulated respiration of skinned fibers increased by 33% (P < 0.05) and 100% (P < 0.001) in sPLA and dPLA, respectively. The maximal ADP-stimulated respiration was 57% (P < 0.001) and 32% (P < 0.01) higher in active rats than in sedentary rats (n = 8), in sPLA and dPLA, respectively. This finding was consistent with a 72% increase in the CS activity in plantaris muscle of exercising rats (P < 0.01). Voluntary running induced a 334% increase in the apparent Km for ADP in sPLA (P < 0.001), and a 61% increase in dPLA (P < 0.05), showing a lower affinity for cytosolic ADP of mitochondria present in both, predominantly glycolytic, and oxidative fibers. There was an increase in the creatine kinase efficacy in both sPLA and dPLA (131%, 75%, P < 0.001, respectively), consistent with an increase in the activity of the mitochondrial isoform of creatine kinase (106%, P < 0.01). It is concluded that, in addition to the well-known increased oxidative capacity, voluntary running is associated with changes in the regulation of oxidative phosphorylation by phosphate acceptors, in both glycolytic and oxidative fibers, in the direction of increased coupling between energy production and energy utilization.     

Long-term repeatability of force, endurance time and muscle activity during isometric contractions.

We determined the repeatability and correlations between force, endurance and muscle activity during isometric contractions over three years. Twenty-six subjects, with and without complaints of the shoulder and neck, performed standardized maximal and submaximal shoulder-abduction contractions and wrist extension-contractions at yearly intervals from 1997 to 1999. Peak forces developed during maximal contraction and the endurance times of submaximal contractions during shoulder abduction and wrist extension were measured. Electromyography (EMG) of muscle activity was recorded bilaterally from the upper trapezius, middle deltoid, and forearm extensor muscles. Root mean square EMG amplitudes were calculated. We found statistically significant associations between peak forces developed during wrist extension and shoulder abduction, and between endurance times of submaximal wrist extension and shoulder abduction. No statistically significant changes in peak force and EMG(peak) were found over the measurement years. The responses were not statistically significantly influenced by gender, or neck and shoulder pain. However, we observed considerable intra-individual variation in the inter-year measurements particularly for the responses to submaximal contraction. Such large variations represent a challenge when attempting to use the responses to interpret the effects of therapies.     

Adaptation to salinity by fish: Alterations in energy transducing status of muscle mitochondria

The functional status of fish muscle mitochondria during exposure of the organism to salinity stress was studied. No alterations were observed in the substrate oxidation capacity. However, the mitochondria appear to be in an uncoupled state during the first few days of exposure and recover thereafter. This could be correlated to high endogenous Ca²⁺ levels in the mitochondria in the early period. A Na⁺-Ca²⁺ antiport in these mitochondria is shown. Evidence is also presented to show that the mitochondria isolated during the early stages of stress are in a swollen state and are unable to contract on addition of ATP and Mg²⁺ Continued exposure to the stress, however, reverses the situation. Publication No. 034 from the Department of Biochemistry.     

Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.     

Lack of effect of nitric oxide on KCl, acetylcholine and substance P induced contractions in ileal longitudinal muscle of the rat

The aim of this study was to determine whether an excess of nitric oxide (NO) (mimicked by addition of NO donors) might produce by itself changes in the contractile responses to acetylcholine (ACh), substance P (SP) and KCl in the longitudinal muscle of the rat ileum. We also studied the calcium handling properties of this tissue in presence of NO donors. The NO donors assayed sodium nitroprusside (SNP) and 3-morpholinosydnonimine hydrochloride (SIN-1), induced different responses. SNP caused an immediate contraction followed by a sustained relaxation, whereas SIN-1 induced an immediate relaxation followed by a contraction. Even after prolonged incubations (up to 90 min), the NO donors SNP and SIN-1 were unable to modify the ACh- and SP-concentration-response curves, as well as the response to 30 mM KCl. The nifedipine-resistant component of the ACh-induced contraction was not modified in presence of SNP. Cyclopiazonic acid (CPA) induced a contraction that was not modified when the tissue was pre-incubated with SNP. Nifedipine caused a sharp relaxation when added during the CPA-induced contraction and, when added previously, it reduced the CPA-induced contractile response. It is concluded that NO excess is not, by itself, responsible for the altered responses to KCl. ACh and SP. The contractility changes observed in the longitudinal muscle of the rat ileum during inflammation could rather be related to the presence of other inflammatory mediators.     

Growth,reproductive function,and tolerance to load tests in rats under repetitive adverse conditions

Wistar rats were subjected to a 3.5-day water deprivation once a month in the period of 1 to 10 months of age. The rats' adaptive abilities proved sufficient for compensation of adverse effects, the rats preserved their normal motor activity, emotionality, and orienting behaviour in the intervals between the effects. In the reproductive period, the males manifested a normal sexual behaviour and fertilizing capacity of their spermatozoids. In the end of the experiment, the experimental animals did not differ from the control animals.     

Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse

The main purpose of this study was to investigate whether consumption of diets enriched in antioxidants attenuates the level of oxidative stress in the senescence-accelerated mouse (SAM). In separate and independent studies, two different dietary mixtures, one enriched with vitamin E, vitamin C, L-carnitine, and lipoic acid (Diet I) and another diet including vitamins E and C and 13 additional ingredients containing micronutrients with bioflavonoids, polyphenols, and carotenoids (Diet II), were fed for 8 and 10 months, respectively. The amounts of glutathione (GSH) and glutathione disulfides (GSSG) and GSH:GSSG ratios were determined in plasma, tissue homogenates, and mitochondria isolated from five different tissues of SAM (P8) mice. Both diets had a reductive effect in plasma; however Diet I had relatively little effect on the glutathione redox status in tissue homogenates or mitochondria. Remarkably, Diet II caused a large increase in the amount of glutathione and a marked reductive shift in glutathione redox state in mitochondria. Overall, the effects of Diet II were tissue and gender specific. Results indicated that the glutathione redox state in mitochondria and tissues can be altered by supplemental intake of a relatively complex mixture of dietary antioxidants that contains substances known to induce phase 2 enzymes, glutathione, and antioxidant defenses. Whether corresponding attenuations occur in age-associated deleterious changes in physiological functions or life span remains unknown.     

Modulation of activity in starling cochlear ganglion units by middle-ear muscle contractions,perilymph movements and lagena stimuli

In the present study three groups of cochlear ganglion neurons were detected which differed in respect to their tone-evoked and spontaneous activity: auditory units which showed an irregular spontaneous discharge, non-auditory neurones with regular activity and such with an irregular spontaneous discharge pattern. Electrically-elicited contractions of the middle-ear muscle influenced the tone-evoked and/or the spontaneous activity of the auditory and the non-auditory neurones with irregular spontaneous discharge but not, however, the regularly firing units. Similar results were obtained with imposed perilymph movements in the cochlea (evoked via the vestibular system. Fractions of all three groups of cochlear ganglion neurones were responsive to direct deformations of the membraneous lagena. Several (auditory and non-auditory) units with irregular discharge were excited during a basilar membrane displacement towards scala vestibuli whereas a basilar membrane motion towards scala tympani resulted in a decrease of the discharge rate. A few units showed a different reaction. The results provide evidence that the neurones with periodic spontaneous discharge innervate the lagena and that this sense organ has no auditory significance in birds. The peripheral origin of the 'non-auditory' neurones with irregular spontaneous activity remains undecided and might be the macula lagenae or the apical portion of the basilar papilla.     

Biochemical adaptation of mitochondria,muscle, and whole-animal respiration to endurance training

The experimental intervention of exercise training has been used to study mitochondrial biosynthesis, and the physiologic integration of subcellular, cellular, and whole-animal energetics. Gross mitochondrial composition was unchanged in rat muscle by a 10-week program of endurance treadmill running. The mitochondrial concentration of iron-sulfur clusters, cytochromes, flavoprotein, dehydrogenases, oxidases, and membrane protein and lipid, as well as the ratios of each component to the others, maintained constant proportions. The mitochondrial content of muscle, however, increased by approximately 100% as did absolute tissue oxidative capacity. The soluble portions of mitochondria maintained a constant total protein content and mass, relative to the membrane fraction, despite adaptive changes in the specific activities of some citric acid-cycle enzymes. Mitochondria from endurance-trained muscles generated normal transmembrane potentials, ADP/O ratios, and respiratory control ratios. Muscle oxidase activity was highly correlated (r = 0.92) with endurance capacity, which increased 403%. Whole-animal maximal O₂ consumption (), however, increased only 14% and was a relatively poor predictor of endurance. Thus, mitochondrial factors, rather than , must play an important role in dictating the limits of endurance activity. Conversely, was strongly related to the maximal intensity of work which could be attained aerobically (r = 0.82). Comparison of O₂ consumption at the mitochondrial, muscle, and whole-animal levels revealed that maximal muscle oxidase activity was not an absolute limitation to : It is concluded that other factors intervene to control the percentage of muscle O₂ consumption capacity which may be utilized during exercise.