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The H55N polymorphism in the Cs gene of A/J mice has been linked to low activity of the enzyme in skeletal muscles. The aim of the study was to test this hypothesis and examine effects of low citrate synthase (CS) activity on palmitate metabolism in muscle cells. Results of the study showed that carriers of the wild type (WT) Cs (C57BL/6J and Balb/cByJ mouse strains) had higher CS activity (p < 0.01) than carriers of the A/J variant (B6.A-(rs3676616-D10Utsw1)/KjnB6 and A/J mouse strains) in the heart, liver and gastrocnemius muscle. Furthermore, the recombinant CS protein of WT showed higher CS activity than the A/J variant. In C2C12 muscle cells the shRNA mediated 47% knockdown of CS activity reduced the rate of fatty acid oxidation compared to the control cells. In summary, our results are consistent with the hypothesis that H55N substitution causes a reduction in CS activity. Furthermore, low CS activity interferes with metabolic flexibility of muscle cells.

Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from dysferlinopathy (Dysfy), polymyositis (PM), and distal myopathy with rimmed vacuoles (DMRV) displayed morphological and biochemical evidences of mitochondrial dysfunction. Proteomic analysis revealed down-regulation of electron transport chain (ETC) subunits, assembly factors, and tricarboxylic acid (TCA) cycle enzymes, with 80 proteins common among the three pathologies. Mitochondrial proteins from muscle pathologies also displayed higher Trp oxidation that could alter the local structure. Cover image for this issue: doi: 10.1111/jnc.13324., (© 2016 International Society for Neurochemistry.)

Owing to the paucity of data on the red muscle of deep-sea fishes, the goal of this study was to determine the proportions of red muscle in demersal fishes and its enzymatic activities to characterize how routine swimming abilities change with depths of occurrence. Cross sectional analysis of the trunk musculature was used to evaluate the proportion of red muscle in 38 species of Californian demersal fishes living at depths between 100 and 3000 m. The activity of metabolic enzymes was also assayed in a sub-set of 18 species. Benthic fishes had lower proportions of red muscle and lower metabolic enzyme activities than benthopelagic species. Mean proportion of red muscle declined significantly with depth with the greatest range of values in shallow waters and species with low proportions found at all depths. This suggested that while sedentary species occur at all depths, the most active species occur in shallow waters. Citrate synthase activity declined significantly with depth across all species, indicating that the mass-specific metabolic capacity of red muscle is lower in deep-sea species. These patterns may be explained by coupling of red and white muscle physiologies, a decrease in physical energy of the environment with depth or by the prevalence of anguilliform body forms and swimming modes in deep-living species., (© 2013 The Fisheries Society of the British Isles.)

There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic-endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity.

Honeybees, Apis mellifera, who show temporal polyethism, begin their adult life performing tasks inside the hive (hive bees) and then switch to foraging when they are about 2-3 weeks old (foragers). Usually hive tasks require little or no flying, whereas foraging involves flying for several hours a day and carrying heavy loads of nectar and pollen. Flight muscles are particularly plastic organs that can respond to use and disuse, and accordingly it would be expected that adjustments in flight muscle metabolism occur throughout a bee's life. We thus investigated changes in lifetime flight metabolic rate and flight muscle biochemistry of differently aged hive bees and of foragers with varying foraging experience. Rapid increases in flight metabolic rates early in life coincided with a switch in troponin T isoforms and increases in flight muscle maximal activities (V (max)) of the enzymes citrate synthase, cytochrome c oxidase, hexokinase, phosphofructokinase, and pyruvate kinase. However, further increases in flight metabolic rate in experienced foragers occurred without additional changes in the in vitro V (max) of these flight muscle metabolic enzymes. Estimates of in vivo flux (v) compared to maximum flux of each enzyme in vitro (fractional velocity, v/V (max)) suggest that most enzymes operate at a higher fraction of V (max) in mature foragers compared to young hive bees. Our results indicate that honeybees develop most of their flight muscle metabolic machinery early in life. Any further increases in flight metabolism with age or foraging experience are most likely achieved by operating metabolic enzymes closer to their maximal flux capacity.

The decline of cellular and especially mitochondrial functions with age is, among other causes, held responsible for a decrease in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10 years) from the Isle of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the change in physiology with age and size might result in a changed escape response behaviour towards predators.

The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

The influence of body size on metabolic rate, muscle enzyme activities and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. We studied two pairs of sister species of centrarchid fish: black bass (largemouth bass Micropterus salmoides and smallmouth bass Micropterus dolomieui) and sunfish (pumpkinseed Lepomis gibbosus and bluegill Lepomis macrochirus). Our goal was to assess the regulatory basis of both intraspecific and interspecific variation relative to body size, as well as to gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from (+0.87 to +0.96). However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19, whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes.

During their fall migration from the Arctic to South America, semipalmated sandpipers Calidris pusilla stop in the Bay of Fundy (east coast of Canada) before flying non-stop for approximately 4500 km across the ocean. Refueling birds double their body mass by feeding on Corophium volutator, an amphipod containing high amounts of n-3 polyunsaturated fatty acids (n-3 PUFA), particularly eicosapentaenoic (20:5) and docosahexaenoic acid (22:6). In mammals, high dietary intake of n-3 PUFA is known to increase capacity for oxidative metabolism. Therefore, we hypothesized that tissue incorporation of n-3 PUFA would be associated with increases in the activity of key muscle enzymes to upregulate energy metabolism for prolonged exercise. Birds were collected at various stages of fat loading to monitor changes in lipid composition and flight muscle enzymes simultaneously. Enzymes were measured to assess oxidative capacity [citrate synthase (CS)], beta-oxidation [carnitine palmitoyl transferase (CPT) and 3-hydroxyacyl dehydrogenase (HOAD)] and glycolytic capacity [lactate dehydrogenase (LDH)]. Changes in the fatty acid composition of muscle membranes (phospholipids) and fuel reserves (neutral lipids) were measured separately to distinguish between membrane-related and systemic effects of n-3 PUFA. Results show that muscle CS and HOAD are stimulated during refueling and that their activities are correlated with n-3 PUFA content in phospholipids (22:6 for CS, 20:5 for HOAD) and in neutral lipids (20:5 for CS). This suggests that 20:5 and 22:6 have different effects on energy metabolism and that they act via changes in membrane structure and systemic mechanisms. CPT and LDH did not change during refueling, but LDH activity was significantly related to the n-3 PUFA content of fuel reserves. This study shows that oxidative capacity increases rapidly during refueling and supports the idea that dietary n-3 PUFA are used as molecular signals to prime flight muscles of some long-distance migrants for extreme exercise.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Honeybees, Apis mellifera, gradually increase their rate of forage uptake as they gain foraging experience. This increase in foraging performance has been proposed to occur as a result of learning; however, factors affecting flight ability such as changes in physiological components of flight metabolism could also contribute to this pattern. Thus, the purpose of this study was to assess the contribution of physiological changes to the increase in honeybee foraging performance. We investigated aspects of honeybee flight muscle biochemistry throughout the adult life, from non-foraging hive bees, through young and mature foragers, to old foragers near the end of their lifespan. Two-dimensional gel proteomic analysis on honeybee thorax muscle revealed an increase in several proteins from hive bees to mature foragers including troponin T 10a, aldolase and superoxide dismutase. By contrast, the activities (V(max)) of enzymes involved in aerobic performance, phosphofructokinase, hexokinase, pyruvate kinase and cytochrome c oxidase, did not increase in the flight muscles of hive bees, young foragers, mature foragers and old foragers. However, citrate synthase activity was found to increase with foraging experience. Hence, our results suggest plasticity in both structural and metabolic components of flight muscles with foraging experience.

Background: Myopathy, probably caused by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in skeletal muscle, rarely occurs in patients taking statins. This study was designed to assess the effect of high-dose statin treatment on cholesterol and ubiquinone metabolism and mitochondrial function in human skeletal muscle., Methods: Forty-eight patients with hypercholesterolemia (33 men and 15 women) were randomly assigned to receive 80 mg/d of simvastatin (n = 16), 40 mg/d of atorvastatin (n = 16), or placebo (n = 16) for 8 weeks. Plasma samples and muscle biopsy specimens were obtained at baseline and at the end of the follow-up., Results: The ratio of plasma lathosterol to cholesterol, a marker of endogenous cholesterol synthesis, decreased significantly by 66% in both statin groups. Muscle campesterol concentrations increased from 21.1 +/- 7.1 nmol/g to 41.2 +/- 27.0 nmol/g in the simvastatin group and from 22.6 +/- 8.6 nmol/g to 40.0 +/- 18.7 nmol/g in the atorvastatin group (P = .005, repeated-measurements ANOVA). The muscle ubiquinone concentration was reduced significantly from 39.7 +/- 13.6 nmol/g to 26.4 +/- 7.9 nmol/g (P = .031, repeated-measurements ANOVA) in the simvastatin group, but no reduction was observed in the atorvastatin or placebo group. Respiratory chain enzyme activities were assessed in 6 patients taking simvastatin with markedly reduced muscle ubiquinone and in matched subjects selected from the atorvastatin (n = 6) and placebo (n = 6) groups. Respiratory chain enzyme and citrate synthase activities were reduced in the patients taking simvastatin., Conclusions: High-dose statin treatment leads to changes in the skeletal muscle sterol metabolism. Furthermore, aggressive statin treatment may affect mitochondrial volume.

Changes in metabolic enzyme activities were examined in three species of pikas that occur over a range of altitudes. Because these closely related mammals live in comparable ecosystems and face similar environmental factors regardless of altitude, modifications of metabolic machinery are probably due to differences in oxygen availability. Citrate synthase (CS), beta-hydroxyacyl CoA dehydrogenase (HOAD) and lactate dehydrogenase (LDH) activities were measured in heart, diaphragm, vastus lateralis, gastrocnemius and soleus muscles. Additionally, the activity levels of both M-LDH (skeletal muscle type) and H-LDH (heart type) isozymes were quantified in tissue samples. Pikas from high altitude had greater CS and HOAD activities in heart and diaphragm when compared with pikas from low altitude, while activity levels did not differ in skeletal muscles. The increase in oxidative enzyme activities in tissues with high metabolic demand is thought to enhance oxygen utilization when oxygen availability is low and may reflect greater metabolic demand on heart and diaphragm tissue. Pikas from high altitude were also found to have greater total LDH activities in all tissues examined. High altitude animals had dramatically higher H-LDH activity (2.3-3.8 times greater) while M-LDH activity was more comparable (1.8 times lower to 1.7 times greater) when compared with low altitude animals. High total LDH activity enables pikas to perform short bouts of anaerobic activity, while high levels of H-LDH isozymes may serve to enhance lactate removal and decrease recovery time in animals living at high altitude.

Reptiles living in heterogeneous thermal environments are often thought to show behavioural thermoregulation or to become inactive when environmental conditions prevent the achievement of preferred body temperatures. By contrast, thermally homogeneous environments preclude behavioural thermoregulation, and ectotherms inhabiting these environments (particularly fish in which branchial respiration requires body temperature to follow water temperature) modify their biochemical capacities in response to long-term seasonal temperature fluctuations. Reptiles may also be active at seasonally varying body temperatures and could, therefore, gain selective advantages from regulating biochemical capacities. Hence, we tested the hypothesis that a reptile (the American alligator Alligator mississippiensis) that experiences pronounced seasonal fluctuations in body temperature will show seasonal acclimatisation in the activity of its metabolic enzymes. We measured body temperatures of alligators in the wild in winter and summer (N=7 alligators in each season), and we collected muscle samples from wild alligators (N=31 in each season) for analysis of metabolic enzyme activity (lactate dehydrogenase, citrate synthase and cytochrome c oxidase). There were significant differences in mean daily body temperatures between winter (15.66+/-0.43 degrees C; mean +/- S.E.M.) and summer (29.34+/-0.21 degrees C), and daily body temperatures fluctuated significantly more in winter compared with summer. Alligators compensated for lower winter temperatures by increasing enzyme activities, and the activities of cytochrome c oxidase and lactate dehydrogenase were significantly greater in winter compared with summer at all assay temperatures. The activity of citrate synthase was significantly greater in the winter samples at the winter body temperature (15 degrees C) but not at the summer body temperature (30 degrees C). The thermal sensitivity (Q(10)) of mitochondrial enzymes decreased significantly in winter compared with in summer. The activity of mitochondrial enzymes was significantly greater in males than in females, but there were no differences between sexes for lactate dehydrogenase activity. The differences between sexes could be the result of the sex-specific seasonal demands for locomotor performance. Our data indicate that biochemical acclimatisation is important in thermoregulation of reptiles and that it is not sufficient to base conclusions about their thermoregulatory ability entirely on behavioural patterns.

This study evaluated the link between swimming endurance and condition of Atlantic cod Gadus morhua that had been fed or starved during the 16 weeks preceding the tests, and assessed whether muscle metabolic capacities explain such links. The condition factor [(somatic mass x fork length(-3))x100] of starved cod was 0.54+/-0.1 whereas that of fed cod was 0.81+/-0.1. In white and red muscle, we measured four glycolytic enzymes: phosphofructokinase (PFK), pyruvate kinase (PK), creatine kinase (CK) and lactate dehydrogenase (LDH), two mitochondrial enzymes: cytochrome c oxidase (CCO) and citrate synthase (CS), a biosynthetic enzyme, nucleoside diphosphate kinase (NDPK), glycogen and protein levels and water content. Muscle samples were taken at three positions along the length of the fish; starvation affected the metabolic capacities of white muscle more than those of red muscle. The levels of glycolytic enzymes and glycogen changed more in white than red muscle during starvation. Both in fed and starved cod, muscle metabolic capacities varied with position along the fish; starvation reduced this longitudinal variation more in white than red muscle. In white muscle of fed cod, the glycolytic enzyme levels increased from head to tail, while in starved cod this longitudinal variation disappeared. In red muscle mitochondrial enzyme levels were highest in the caudal sample, but fewer differences were found for glycolytic enzymes. Swimming endurance was markedly affected by fish condition, with starved fish swimming only 30% of the time (and distance) of fed fish. This endurance was closely linked with the number of burst-coast movements during the test and the activity of CCO and LDH in white muscle. The number of burst-coast movements was significantly linked with condition factor and PFK activity in caudal red muscle and gill arch mass. Our data indicated that cod use both glycolytic and oxidative capacities to support endurance swimming. Furthermore, swimming endurance is linked with the metabolic capacities of red and white muscle.

It had been suggested that the activity of anaerobic enzymes in the white muscle of fish increases exponentially with body size to meet the increasing hydrodynamic costs of burst swimming. We tested whether this relationship holds across a very large size range of striped bass, spanning a nearly 3,000-fold range in body mass. We examined the scaling of marker enzymes of anaerobic (lactate dehydrogenase and pyruvate kinase) and aerobic (citrate synthase and malate dehydrogenase) metabolism in the red and white locomotor muscles. In white muscle, we found positive scaling of anaerobic enzymes only in smaller fishes. Positive scaling of anaerobic enzymes was not found among the samples that included fishes >1,000 g despite having a sufficiently large sample size to detect such scaling. The absence of positive scaling in the white muscles of large bass suggests that they are unable to generate sufficient power to sustain relative burst swimming performance. Enzymes from aerobic pathways had activities that were mass independent in both red and white muscle. Red and white muscles were metabolically distinct except among the smallest fishes. Among young of the year, the anaerobic capacity of red muscle approached that of white muscle and also showed positive scaling. This unusual pattern suggests that red muscle might augment white muscle during burst swimming and add to the total power generated by these small fish. Maximizing burst swimming performance may be critical for small fishes vulnerable to predation but unimportant for large fishes.

Mitochondrial oxidative metabolism was examined in two infants with Pompe's disease. The clinical diagnosis was confirmed by the demonstration of intralysosomal glycogen accumulation and a deficiency of acid alpha-D-glucosidase in muscle biopsies. Light and electron microscopy studies demonstrated a normal number of mitochondria with normal ultrastructure. Spectrophotometric measurements revealed that the specific activities of citrate synthase and the partial reactions of electron transport were markedly elevated in the skeletal muscle homogenates prepared from both infants with Pompe's disease when calculated as micromoles per minute per gram wet weight of tissue. However, when respiratory chain enzyme activities were expressed relative to citrate synthase as a marker mitochondrial enzyme, a different pattern emerged, in which all Pompe muscle respiratory enzymes, except complex IV, were decreased relative to control subjects. These observations demonstrate that caution should be exercised when analyzing and interpreting data obtained from tissue homogenates in general and, in particular, in those prepared from tissues in which the wet weight of tissue may be altered, for example, by pathologic accumulation of carbohydrate or lipid.

The objectives of this study were to determine the effects of 10 consecutive days of moderate-intensity training on 1) the muscular metabolic response to exercise at 100% of the pre-training maximum rate of oxygen consumption (VO2max); and 2) mitochondrial enzyme markers (citrate synthase, CS; succinate dehydrogenase, SDH; 3-hydroxy-acyl-CoA dehydrogenase, HAD) of oxidative capacity in middle gluteal muscle. Six mature, unfit Thoroughbred horses completed both incremental (for determination of VO2max) and high-intensity exercise protocols before (HI1) and after (HI2) training. Training consisted of 10 consecutive days of running at 55% VO2max for 60 min per day (13-14 km/day). For the HI, horses completed a 10 min warm-up, followed by exercise at 100% of pre-training VO2max (mean speed 9.8 m/s) until fatigue. Training resulted in an 8.9% increases in VO2max (Pre: 142 +/- 4 ml/kg bwt/min; Post: 155 +/- 4 ml/kg bwt/min) and a 24% increase in run time to fatigue during HI. Whereas VO2 during HI was not altered by training, peak values for VCO2 and R were significantly lower following training. Compared to HI1, there was a 45% reduction in the net rate of muscle glycogenolysis during HI2. Peak (end exercise) values for plasma and muscle lactate concentrations decreased by 22 and 23%, respectively, after training. Training also attenuated the exercise-associated increase in plasma norepinephrine, but there was no effect on plasma epinephrine concentrations. Maximal activities of CS, SDH, and HAD were unaltered by training. We conclude that 10 days of moderate-intensity exercise results in decreases in muscle glycogenolysis and anaerobic metabolism during high-intensity exercise at the same absolute workload. Furthermore, development of measurable increases in mitochondrial oxidative potential may not be required for expression of these metabolic adaptations in early training.

To investigate temporal changes in capillarization and increases in mitochondrial enzyme activity, rabbit tibialis anterior muscles underwent chronic low-frequency stimulation for up to 50 days. Capillary density (CD), capillary-to-fiber ratio (C/F), intercapillary distance (ICD), and mean capillary area (MCA), as well as several other parameters of capillarization, were examined. In addition, tissue levels of mRNA specific to vascular endothelial growth factor (VEGF) were assessed by reverse transcriptase-polymerase chain reaction. Citrate synthase (CS) activity, a marker of aerobic-oxidative metabolic potential, was measured in the same muscles. Significant increases in CD and C/F, respectively, and decreases in ICD and MCA were observed after 2 days. These changes reached stable maxima by 14 days. The increases in capillarization occurred in a fiber-type-specific manner, affecting type IId fibers before types IIda and IIa. VEGF mRNA levels increased in a bimodal time pattern with a first elevation (2.5-fold) after 1 day and a second (9-fold) after 6-8 days. Increases in CS were first noted after 8 days. Obviously, increases in capillarization as induced by enhanced contractile activity precede increases in the aerobic-oxidative potential of energy metabolism.

Twelve pairs of healthy sedentary males matched for their body mass index (BMI) with either a low insulin response (LIR; a stage of prediabetes) or a high (HIR; controls) to a standardized glucose infusion test (GIT) were studied in respect to their exercise capacities (W(OBLA), W(SL) and relative W(OBLA):W(OBLA) x W(SL)(-10 x 100), muscle fiber composition (%ST), muscle citrate synthase activity (CS), muscle ubiquinone (MUQ), MUQ over %ST (muscle quality index, MQI), and peripheral insulin sensitivity (PIS) as described with insulin-clamp techniques (SIGITmean). LIR and HIR displayed normal PIS and positive relationships versus exercise capacity. LIR's but not HIR's relative W(OBLA) was related to CS as earlier only documented in endurance athletes but at a lower level than in athletes. This pointed at a poor peripheral oxygen delivery in LIR. LIR's MQI decreased relative to HIR's the higher the muscle CS indicating radical related muscle trauma in LIR as in athletes. LIR representing prediabetes described muscle anomalies, which could represent prestages of the lesions observed in type-2 diabetes. They are claimed to be responsible for insulin-, glucose-, lipid-resistance, and peripheral circulatory resistance.

Flight muscles of the cricket Gryllus firmus are polymorphic, existing as pink or white phenotypes. White muscles are smaller in size, have reduced number and size of muscle fibers, and have reduced in vitro enzyme activities and respiration rates relative to pink muscles of newly molted, fully winged adults. G. firmus is also polymorphic for wing length. All newly molted long-winged adults exhibited the pink-muscle phenotype, while most newly molted short-winged adults exhibited the white-muscle phenotype, which resulted from arrested muscle growth. As long-winged adults aged, fully grown pink muscle was transformed into white muscle via histolysis. The substantially higher respiration rate of pink muscle likely contributes to the elevated whole-organism respiration rate of long-winged females, which has been documented previously and which is thought to divert nutrients from egg production. Histolyzed white flight muscle from long-winged crickets also exhibited significantly elevated respiration rate and enzyme activities compared with underdeveloped white muscle from short-winged adults, although these differences were not as great as those between pink and white muscles. Fecundity was much more elevated in females with white verus pink flight muscles than it was in females with short versus long wings. The fitness gain resulting from flightlessness has typically been estimated in previous studies by comparing enhanced egg production of short-winged and long-winged females, without considering the influence of flight-muscle variation. Our results suggest that the magnitude of this fitness gain has been substantially underestimated.

We examined skeletal and cardiac muscle responses of mature (8 mo) and old (23 mo) male Fischer 344 rats to 14 days of hindlimb suspension. Hexokinase (HK) and citrate synthase (CS) activities and GLUT-4 glucose transporter protein level, which are coregulated in many instances of altered neuromuscular activity, were analyzed in soleus (Sol), plantaris (PI), tibialis anterior (TA), extensor digitorum longus (EDL), and left ventricle. Protein content was significantly (P < 0.05) lower in all four hindlimb muscles after suspension compared with controls in both mature (21-44%) and old (17-43%) rats. Old rats exhibited significantly lower CS activities than mature rats for the Sol, Pl, and TA. HK activities were significantly lower in the old rats for the Pl (19%) and TA (33%), and GLUT-4 levels were lower in the old rats for the TA (38%) and EDL (24%) compared with the mature rats. Old age was also associated with a decrease in CS activity (12%) and an increase in HK activity (14%) in cardiac muscle. CS activities were lower in the Sol (20%) and EDL (18%) muscles from mature suspended rats and in the Sol (25%), Pl (27%), and EDL (25%) muscles from old suspended rats compared with corresponding controls. However, suspension was associated with significantly higher HK activities for all four hindlimb muscles examined, in both old (16-57%) and mature (10-43%) rats, and higher GLUT-4 concentrations in the TA muscles of the old rats (68%) but not the mature rats. These results indicate that old age is associated with decreased CS and HK activities and GLUT-4 protein concentration for several rat hindlimb muscles, and these variables are not coregulated during suspension. Finally, old rat skeletal muscle appears to respond to suspension to a similar or greater degree than mature rat muscle responds.

The metabolic activities of skeletal muscles were studied in male rats exposed to hypobaric-hypoxia at about 550 Torr for 8 h per day for 2 weeks. Rats were divided into three groups; control (normoxic control), diurnal hypoxic (DH) exposure, and nocturnal hypoxic (NH) exposure groups. The changes in body weight and daily diet intake of the NH group were lower than the other groups (p < 0.01). The weights of fat in the abdominal cavity of both NH and DH groups were less than that of the control group. The red blood cell count, hemoglobin concentration, and hematocrit values were significantly increased in the hypoxic groups. The plasma glucose level in the NH group was significantly less than the control group (p < 0.05). The lactate dehydrogenase/citrate synthase (LDH/CS) activity ratios in the skeletal muscle tended to be lower in both hypoxic groups than in the control group. The swimming times to exhaustion at mild and high intensities that were measured after 2 weeks, loaded with a weight equivalent to 2.5% of the body weight, improved in the DH group. There were insignificant differences in the metabolic activity of skeletal muscles and blood characteristics between the NH and DH groups, but endurance swimming times in the DH group tended to be improved as compared to the NH group. We conclude that the DH group became competent in endurance work, which is believed to be driven from the combined effects of increased O2 transport capacity of the blood and enhanced O2 utilization capability by mitochondrial enzyme activity.

We examined whether muscle contractile activity directly modulates GLUT4 protein content in rat skeletal muscle without the participation of insulin action or via amplified insulin action. To attain this purpose, the effects of increased, by training, or eliminated, by denervation, muscle contractile activity on muscle GLUT4 protein concentration were investigated in severely insulin-deficient diabetic rats. For the first set of experiments, insulin-deficient diabetic rats (induced by injection of 80 mg/kg B.W. streptozotocin) were trained for three weeks by treadmill running (90 min/day, 19 m/min, 10%, 6 days/week). GLUT4 protein concentration in soleus muscle was increased by 48% (p < 0.01) as compared with diabetic sedentary animals. For the second set of experiments, rats were injected with streptozotocin (100 mg/kg). The muscles innervated by the sciatic nerve of one leg were denervated four days after injection of streptozotocin. Three days after denervation, soleus muscles in both legs were excised. Insulin deficiency decreased GLUT4 protein concentration in innervated soleus muscle. In insulin-deficient diabetic rats, denervation also decreased soleus GLUT4 protein concentration by 50% (p < 0.01) as compared with the contralateral innervated muscle. Furthermore, the effects of insulin-deficiency and denervation on GLUT4 protein concentration were additive. These results provide evidence that muscle contractile activity directly modulates skeletal muscle GLUT4 protein concentration independent of insulin action.

The interrelationships among glucose uptake, GLUT-4 protein, and citrate synthase activity in insulin-resistant skeletal muscle were investigated. Female obese (fa/fa) Zucker rats were randomly assigned to treadmill training, ingestion of the selective beta 2-adrenergic agonist clenbuterol, or sedentary control groups. After 7-8 wk of treatment, hindlimbs were perfused to determine maximal insulin-stimulated (10 mU/ml) 2-[3H]deoxy-D-glucose (2-DG) uptake. Exercise training significantly enhanced 2-DG uptake and GLUT-4 protein in red gastrocnemius and plantaris. Alternatively, 2-DG uptake was not altered in soleus after exercise training despite a 52% increase in GLUT-4 protein. The increases in GLUT-4 protein in red gastrocnemius, plantaris, and soleus of the trained rats were accompanied by increases in citrate synthase activity. In contrast to exercise training, clenbuterol administration decreased citrate synthase activity in red and white gastrocnemius, yet had no effect on GLUT-4 protein levels or maximal insulin-stimulated 2-DG uptake. Clenbuterol treatment did, however, increase citrate synthase activity and GLUT-4 protein in soleus. These findings indicate that total GLUT-4 protein largely determines the maximal rate of insulin-stimulated glucose uptake in fast-twitch muscle, whereas in slow-twitch muscle it does not. In addition, the results demonstrate that coordination of proteins governing glucose uptake and disposal may be disrupted in a fiber type-specific manner. Overall, the findings raise important questions as to whether regulation of proteins governing glucose uptake and disposal differs significantly among fiber types.

We examined the effects of short-term (5 weeks) and long-term (12 weeks) physical training on actual and total activities, protein content and mRNA abundance of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle. The actual and total activities were significantly increased approximately 60% and approximately 40%, respectively, by long-term training. No effects of short-term training on activities were observed. The increase in the total activity corresponded to increased protein content of the E1 alpha and E2 components of the complex. On the other hand, mRNA abundance for E1 alpha and E2 were not affected by the training, but that for E1 beta was slightly, but significantly increased by both short-term and long-term trainings. These divergent alterations of the message levels for the subunits of the complex suggest that posttranslational regulatory mechanisms determine the amount of the complex in skeletal muscle. Since the complex is located in the mitochondrial matrix space, mitochondrial biogenesis in response to the training was examined by determining the content of mitochondrial DNA in the muscle. The mitochondrial DNA was proportionally increased with the total activity as well as the protein content of the complex, suggesting that expression of branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle in response to physical training is associated with mitochondrial biogenesis.

Experiments were performed to determine if exercise training reduces collateral or microvascular resistances in the hindlimb of rats with arterial insufficiency. After right femoral arterial ligation (age 10 wk), rats were divided into sedentary (Sed) and treadmill-trained (Tr) groups (7-9 wk, final intensity: 27 m/min, 6 degree grade, 60 min/day). Minimal resistances (mmHg.ml-1.min.100 g) of the total limb (RT), collateral vessels (RC), and the microcirculations distal (Rfmc) and proximal (Rimc) to the ligation site were determined during pump perfusion of the hindlimbs. RT was lower in nonligated (open) and acutely ligated limbs of Tr than Sed rats (open: 0.69 +/- 0.011 vs. 0.93 +/- 0.071; acute: 0.92 +/- 0.028 vs. 1.18 +/- 0.070 mmHg.ml-1.min.100 g) but not in chronically ligated limbs (chronic: 0.88 +/- 0.072 vs. 1.00 +/- 0.046 mmHg.ml-1.min.100 g). RC was similar between the chronically ligated limbs of Sed and Tr rats (1.69 +/- 0.165 vs. 1.97 +/- 0.227 mmHg.ml-1.min.100 g) and was approximately 70% lower than in acutely ligated limbs of both groups. Rfmc and Rimc were not affected by arterial ligation, but Rimc was significantly lower in Tr than in Sed rats (acute: 1.05 +/- 0.026 vs. 1.54 +/- 0.163; chronic: 1.24 +/- 0.071 vs. 1.81 +/- 0.202 mmHg.ml-1.min.100 g). These results indicate that the primary site of vascular adaptations to chronic arterial ligation is in the collateral vessels. Exercise training does not significantly alter the collateralization process but may provide protection against acute arterial occlusion by stimulating microvascular growth.

Muscle carnitine levels were examined in 31 younger [mean (SD), 27 (5) years] and 27 older [49 (8) years] men. Needle biopsies were obtained from the lateral gastrocnemius or vastus lateralis muscles and assayed for free and total carnitine concentrations via a 5,5'-Dithiobis-(2-nitrobenzoic acid) DTNB-linked spectrophotometric procedure. A subgroup of subjects (n = 28) were assessed for citrate synthase (CS) and succinate dehydrogenase (SDH) activity, and type I muscle fiber composition (% type I fibers). An additional sub-group of nine subjects was assessed for free and total serum carnitine levels. No mean (SEM) differences in free [21.6 (0.7) vs 20.3 (0.9) mumol.g dry weight-1] and total [26.4 (0.6) vs 26.1 (0.9) mumol.g dry weight-1) muscle carnitine levels were found between the younger and older subjects, respectively. Correlational data revealed no significant relationships between total muscle carnitine and CS (r = -0.36), SDH (r = -0.26), or % type I fibers (r = -0.16). In addition, there was a low non-significant relationship between serum and muscle total carnitine concentrations (r = -0.44). These findings suggest that muscle carnitine levels are similar between younger and older males, and there does not appear to be any relationship between muscle carnitine and markers of muscle oxidative potential (i.e., oxidative enzymes, % type I fiber). Since serum carnitine is often used as an indicator of body carnitine status, it is noteworthy that we found a low negative relationship between blood and muscle carnitine concentrations.

Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.

The purpose of this study was to determine whether dilation of arterial vessels preceding the microcirculation contributes differentially to increases in skeletal muscle blood flow during contractions in anesthetized sedentary (SED) or trained (TR) rats. Experiments were performed in the spinotrapezius muscle of adult male Sprague-Dawley rats. Before and immediately after muscle contractions (2, 4, or 8 Hz), intravascular pressures, red blood cell velocities, and vessel diameters were measured in terminal feed arteries at a site before penetration into the tissue. Pressure was also measured in the accompanying vein. Contraction-induced changes in vascular resistance were calculated for upstream (Rup), spinotrapezius muscle microvascular (Rst), and downstream segments. At rest, Rup accounted for less (32 vs. 40%) and Rst for more (59 vs. 47%) of total resistance in TR than in SED rats. At 8 Hz, contractions produced significantly greater functional dilation (SED, 138 +/- 14 microns; TR, 178 +/- 12 microns) and hyperemia (SED, 11.9 +/- 3.2 x control; TR, 16.8 +/- 3.1 x control) in TR than in SED rats. Inflow pressures did not change, and outflow pressures increased significantly with contractions. Rup and Rst each decreased 60-80% after 2-Hz contractions and > 90% after 8-Hz contractions. Therefore, feed artery dilation contributes significantly to functional hyperemia in the rat spinotrapezius muscle. Furthermore, it appears that aerobic exercise training results in a redistribution of segmental vascular resistance between feed vessels and the microcirculation.

The effects of L-carnitine on the pyruvate dehydrogenase (PDH) complex and carnitine palmitoyl transferase (CPT) were studied in muscle of 16 long-distance runners (LDR). These subjects received placebo or L-carnitine (2 g orally) during a 4-week period of training. Athletes receiving L-carnitine showed a dramatic increase (P < 0.001) in the PDH complex activities. By contrast, the levels of CPT, both 1 and 2, were unchanged. No significant changes were observed after placebo administration. We previously reported [Huertas R. et al., Biochem. Biophys. Res. Commun. 188 (1992) 102-107] that L-carnitine induces an increase in the activities of complexes I, III and IV of the respiratory chain in muscle of LDR. Taken together, our data suggest that the improvement in (maximal oxygen consumption) VO2max observed in LDR after L-carnitine administration is based on these biochemical findings.

The mitochondrial pyruvate carboxylase catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. Since pyruvate carboxylase generates oxaloacetate for Krebs cycle function, it is proposed that the enzyme activity may be enhanced by exercise. To investigate this proposition, pyruvate carboxylase activity was determined in the heart, soleus and gastrocnemius (white portion) muscles of sedentary and swimming-trained adult rats (1 hour per day, 5 days a week, during 5 weeks) under the following conditions: rest, one hour of exercise and exhaustion. The results show that the pyruvate carboxylase activity is increased during exercise in both the sedentary and trained groups of rats. The stimulatory mechanism is unknown but it is possibly related to the generation of pyruvate from the breakdown of glycogen and acetyl CoA during fatty acid oxidation.

We hypothesized that an exercise training program consisting of treadmill running at 32 m/min up a 15% incline, 90 min/day, 5 days/wk for 12-14 wk, would elicit vascular adaptation in skeletal muscle of all fiber types in rats. This hypothesis was based on previous reports that this intensity and duration of training caused increases in oxidative capacity in rat skeletal muscle of all fiber types. Skeletal muscle vascular transport capacity was examined with measurements of total and regional (radiolabeled microspheres) flow capacity, capillary filtration coefficient (CFC), and permeability-surface area product (PS) for 51Cr-EDTA in maximally vasodilated (papaverine) hindquarters of control (C; n = 25) and exercise-trained (ET; n = 26) rats. CFC was increased in ET (0.038 +/- 0.001 vs. 0.030 +/- 0.001 ml.min-1 x mmHg-1 x 100 g-1; P < or = 0.001). PS was greater in ET than C (7.80 +/- 0.33 vs. 6.39 +/- 0.37 ml.min-1 x 100 g-1; P < or = 0.01). Citrate synthase activity was increased in the soleus (25%; P < or = 0.05), the medial head (35%; P < or = 0.05), and the red portion of the long head (45%; P < or = 0.005) but not in the white portion of the long head of triceps brachii (P = 0.14) of ET rats. Pressure-flow relationships indicate that total flow was greater (P < or = 0.05) in ET hindquarters at all perfusion pressures. Regional flow data revealed that increases in flow capacity were not evident in muscles composed of all fiber types as predicted.(ABSTRACT TRUNCATED AT 250 WORDS)

These experiments examined the influence of exercise intensity and duration on antioxidant enzyme activity in locomotor muscles differing in fiber type composition. Nine groups of female Sprague-Dawley rats (age 120 days) exercised 4 days/wk on a motor-driven treadmill for 10 wk. The impact of three levels of exercise intensity (low, moderate, and high: approximately 55, approximately 65, and approximately 75% of maximal oxygen consumption, respectively) and exercise duration (30, 60, and 90 min/day) was assessed. Sedentary animals served as controls. Oxidative capacity in the soleus and white and red gastrocnemius was assessed by measurement of citrate synthase (CS) activity, and antioxidant capacity was evaluated by assay of total superoxide dismutase, catalase, and total glutathione peroxidase (GPX) activities. In all muscles, CS activity increased as a function of exercise duration. Furthermore, in the soleus and white gastrocnemius, the magnitude of the training-induced increase in CS activity was directly related to exercise intensity. In contrast, the peak increase in CS activity in the red gastrocnemius was relatively independent of exercise intensity. Catalase activity was not increased (P > 0.05) in any muscle with training. Training-induced changes in superoxide dismutase and GPX activities were muscle specific; specifically, exercise training significantly (P < 0.05) increased superoxide dismutase activity in the soleus as a function of exercise duration up to 60 min/day. Conversely, training-induced significant (P < 0.05) increases in GPX activity occurred in red gastrocnemius only; the magnitude of the GPX increase was directly related to exercise duration but relatively independent of intensity. These data demonstrate that exercise training-induced changes in muscle antioxidant enzymes are muscle specific.

Samples were taken at slaughter from heart and both locomotor and nonlocomotor muscles from animals of similar body weight but adapted to different levels of activity: three horses and three steers. All samples were analyzed biochemically to measure the activity of key metabolic enzymes. The skeletal muscles were analyzed histochemically for fibre type composition, fibre area and capillary supply. The general pattern of differences in fibre type composition and metabolic profile between muscle groups was similar in both horses and steers. The hearts of both species had high citrate synthase (CS), 3-OH-acylCoA-dehydrogenase (HAD) and hexokinase (HK) and low lactate dehydrogenase (LDH) activities. In both species, deep portions of muscles and muscles localized deeper in the body had a more oxidative metabolic profile than superficial portions and muscles. Taking all muscles into account, it was found that CS and HAD were higher and LDH lower in horse than in steer muscles. Horse muscles contained more type IIA fibres and had a higher capillary supply than steer muscles. There was no difference between the two species regarding mean fibre area. The adaptation of the horse to a higher level of activity in comparison with steers was reflected in a higher oxidative capacity, better vascularization and a larger proportion of type IIA fibres. It was also obvious from these results that the ATPase fibre-typing system does not reflect the metabolic profile of a muscle.

Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.

A study was conducted, as part of the integrated National Aeronautics and Space Administration Space Life Sciences 1 mission flown in June of 1991, to ascertain the effects of 9 days of exposure to zero gravity on the capacity of rodent skeletal muscle fiber types to oxidize either [14C]pyruvate or [14C]palmitate under state 3 metabolic conditions, i.e., nonlimiting amounts of substrate and cofactors. In addition, activity levels of marker enzymes of the tricarboxylic acid cycle, malate shuttle, and beta-oxidation were measured. Results showed that significant differences in muscle weight occurred in both the predominantly slow vastus intermedius and predominantly fast vastus lateralis of flight vs. control groups (P < 0.05). Total protein content of the muscle samples was similar between groups. Both pyruvate oxidation capacity and the marker oxidative enzymes were not altered in the flight relative to control animals. However, the capacity to oxidize long-chain fatty acids was significantly reduced by 37% in both the high- and low-oxidative regions of the vastus muscle (P < 0.05). Although these findings of a selective reduction in fatty acid oxidation capacity in response to spaceflight are surprising, they are consistent with previous findings showing 1) an increased capacity to take up glucose and upregulate glucose transporter proteins and 2) a marked accumulation of triglycerides in the skeletal muscles of rats subjected to states of unloading. Thus, skeletal muscle of animals exposed to non-weight-bearing environments undergo subcellular transformations that may preferentially bias energy utilization to carbohydrates.

To examine effects of aging and endurance training on human muscle metabolism during exercise, 31P magnetic resonance spectroscopy was used to study the metabolic response to exercise in young (21-33 yr) and older (58-68 yr) untrained and endurance-trained men (n = 6/group). Subjects performed graded plantar flexion exercise with the right leg, with metabolic responses measured using a 31P surface coil placed over the lateral head of the gastrocnemius muscle. Muscle biopsy samples were also obtained for determination of citrate synthase activity. Rate of increase in P(i)-to-phosphocreatine ratio with increasing power output was greater (P < 0.01) in older untrained [0.058 +/- 0.022 (SD) W-1] and trained men (0.042 +/- 0.010 W-1) than in young untrained (0.038 +/- 0.017 W-1) and trained men (0.024 +/- 0.010 W-1). Plantar flexor muscle cross-sectional area and volume (determined using 1H magnetic resonance imaging) were 11-12% (P < 0.05) and 16-18% (P < 0.01) smaller, respectively, in older men. When corrected for this difference in muscle mass, age-related differences in metabolic response to exercise were reduced by approximately 50% but remained significant (P < 0.05). Citrate synthase activity was approximately 20% lower (P < 0.001) in older untrained and trained men than in corresponding young groups and was inversely related to P(i)-phosphocreatine slope (r = -0.63, P < 0.001). Age-related reductions in exercise capacity were associated with an altered muscle metabolic response to exercise, which appeared to be due to smaller muscle mass and lower muscle respiratory capacity of older subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of aerobic exercise training, chronic administration of the selective beta 2-adrenergic agonist clenbuterol, and the combination of these two treatments on muscle insulin resistance were compared in female obese (fa/fa) Zucker rats. Rats were randomly assigned to trained, clenbuterol, clenbuterol-trained, or control groups. Training consisted of treadmill running for 2 h/day at 18 m/min up an 8% grade. Clenbuterol was administered by intubation (0.4-0.8 mg.kg body wt-1 x day-1) approximately 30 min before the rats ran each day. After 8 wk of treatment, muscle insulin resistance was assessed via hindlimb perfusion in the presence of 8 mM glucose and a submaximal (500 microU/ml) insulin concentration. Training increased citrate synthase activity (mumol.g wet wt-1 x min-1) by 32-74% and insulin-stimulated glucose uptake by 45%. Clenbuterol ingestion induced a 17-29% increase in muscle mass but decreased citrate synthase activity by 34-42% and had no effect on muscle glucose uptake. Administration of clenbuterol to rats that exercise trained prevented the training-induced improvement in insulin-stimulated glucose uptake and attenuated the increases in citrate synthase activity. In addition, both clenbuterol-treated groups displayed a 42% decrease in beta-adrenergic receptor density. The results indicate that clenbuterol administration, possibly through beta-adrenergic receptor downregulation, attenuated a cellular reaction essential for the exercise training-induced increase in citrate synthase activity and improvement in skeletal muscle insulin resistance of the obese Zucker rat.

Hindlimb muscle sarcolemmal vesicles were purified from three age-matched groups of female Sprague-Dawley rats: sedentary control (CON; n = 10), sprint trained (ST; n = 8), and endurance trained (ET; n = 9). Membrane isolations from the three groups were not significantly different in protein yield or purification index. Blood lactate concentration was determined in resting CON rats and running ET and ST rats during the final week. Both the ST and ET groups were significantly higher in citrate synthase (vs. CON) in the soleus and mid-vastus lateralis. The time course of 1 mM L-(+)-lactate uptake in vesicles from the three groups showed no significant difference at any of the five time points tested under zero-trans conditions. Saturation kinetics were examined at nine lactate concentrations, and Lineweaver-Burk plots revealed no difference between groups in apparent Michaelis-Menten constant or maximal transport velocity. Vesicles from CON and ET rats were used to investigate cis inhibition of 0.1 mM L-(+)-lactate transport by four unlabeled monocarboxylates: L-(+)-lactate, D-(-)-lactate, pyruvate, and alpha-cyanohydroxycinnamate at 0.1, 1.0, and 10 mM. Under pH gradient-stimulated L-(+)-lactate transport conditions, cis inhibition was affected by neither D-(-)-lactate nor endurance training. We conclude that the lactate transporter has distinct cis-inhibitory specificity, is stereospecific, and is stimulated when confronted with parallel lactate and proton gradients but that spring and endurance training do not alter lactate transport rate or capacity under these conditions.

The aim of this work is to expand the knowledge about fiber distribution, capillarization, and metabolic potential in different anatomic sections of the left and right human lumbar paravertebral muscles and further to investigate whether a relationship between these parameters and trunk muscle endurance exists. Lumbar (L3) paravertebral muscle samples were taken bilaterally from six young male cadavers (study 1) and from 10 normal young healthy men (study 2). The main findings are that the paravertebral muscles are dominated by slow twitch fibers with small fiber areas and a well-developed capillary network. This is most pronounced in the longissimus muscle. Glycogen concentration and enzyme activities (glycogen phosphorylase, lactate dehydrogenase, citric synthetase and 3-hydroxyacyl-CoA-dehydrogenase) in both aerobic and anaerobic metabolic pathways are large compared to other muscles. No obvious bilateral differences in the histochemical and biochemical results were present. The isometrical trunk extensor endurance time is markedly increased compared to other human skeletal muscles.

To test whether repeated HBO exposures would increase activity of skeletal muscle metabolic enzymes, 27 rabbits (3 groups) were exposed 90 min/day, 5 days/wk to either 100% O2 at 243 kPa (HBO), 100% O2 at 101 kPa (HIO), or 21% O2 at 101 kPa (CON). Four animals per group were killed after 2 wk treatment, and the remaining five per group were killed after 8 wk of treatment. Soleus, plantaris, and tibialis anterior muscles were removed, and the activities of adenylate kinase, alpha-glycerophosphate dehydrogenase, and citrate synthase were measured. After 8 wk there was no difference in enzyme activity between groups for either plantaris or tibialis anterior. In the soleus after 8 wk there was no difference between groups in adenylate kinase activity, but alpha-glycerophosphate dehydrogenase activity was 56% greater (P < 0.05) in HBO than in HIO and 50% greater than in CON, and citrate synthase activity in HBO was 24% greater (P < 0.05) than that in HIO and 36% greater than that in CON. Inasmuch as the soleus is a postural muscle, these results suggest that long-term HBO treatments can increase enzyme activity in an actively contracting muscle.

The rates of muscle glucose uptake of trained (TR) and untrained (UT) obese Zucker rats were assessed by hindlimb perfusion under basal conditions (no insulin) in the presence of a maximally stimulating concentration of insulin (10 mU/ml) and after muscle contraction elicited by electrical stimulation of the sciatic nerve. Perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H]glucose. Muscle GLUT-4 concentration was determined by Western blot analysis and expressed as a percentage of a heart standard. The rates of insulin-stimulated glucose uptake were significantly higher in the plantaris, red gastrocnemius (RG), and white gastrocnemius (WG), but not the soleus or extensor digatorum longus (EDL) of TR compared with UT rats. After muscle contraction the rates of glucose uptake in the TR rats were significantly higher in the soleus, plantaris, and RG. TR rats had significantly higher GLUT-4 protein concentration and citrate synthase activity than the UT rats in the soleus, plantaris, RG, and WG. Basal plasma membrane GLUT-4 protein concentration of TR rats was 144% above UT rats (P < 0.01). Stimulation by insulin and contraction resulted in a significant increase in plasma membrane GLUT-4 protein concentration in UT rats only. However, plasma membrane GLUT-4 protein concentration in insulin- and contraction-stimulated TR rats remained 53% and 30% greater than that of UT rats, respectively (P < 0.05). Exercise training did not alter basal, insulin-, or contraction-stimulated GLUT-4 functional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle cell fiber types in gracilis, rectus femoris, and long head of triceps brachii muscles of ferrets and dogs were identified on serial sections stained for myosin ATPase after preincubation at pH values of 9.8, 4.6, and 4.3 and for NADH-tetrazolium reductase (NADH-TR) activity. Although fiber types I and II were identified, the ATPase stain did not demonstrate classic type IIA/IIB fiber differences in either species. However, two type II fiber subtypes could be distinguished in the ferret because they differed slightly in staining intensity with ATPase at pH 4.3 and markedly with NADH-TR. One ferret type II fiber (designated II dark or IID) was smaller, slightly darker on ATPase, more oxidative on NADH-TR, and comprised more muscle volume than the other type II fiber (designated II light IIL). The IID fibers of ferret may represent the IID/X fibers of other authors. Both ferret type II fiber subtypes stained darker at pH 4.3 than canine II fibers. The NADH-TR staining indicated high oxidative activity in canine and ferret type I fibers. In contrast, type II fibers in the dog and IIL fibers in the ferret were moderately oxidative. Canine type IIC fibers were intermediate between type I and type II, whereas in the ferret, type IIC fibers were highly oxidative, as were type IID fibers. Ferret muscles are more oxidative than canine muscles according to NADH-TR staining. Also, ferret muscles possess 40-100% higher citrate synthase activity as compared to canine muscles.

This study compared in vivo measurements of muscle metabolism in humans with magnetic resonance spectroscopy (MRS) and in vitro analysis of biopsies. Healthy subjects [4 young males, 28.2 +/- 6.8 (SD) yr, and 6 older subjects (5 males, 1 female), 66 +/- 6.0 yr] performed a maximal cycle ergometer test, and MRS measurements of the calf muscles and needle biopsies of the lateral gastrocnemius were performed. Biopsies were analyzed for fiber type and citrate synthase (CS) activity. MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ATP, and pH were made using a 1.8-T 78-cm clear-bore magnet-and-spectrometer system. Two or three 5-min bouts of plantar flexion were performed against variable resistance to deplete PCr levels to 50% of resting values (mean end pH 6.99). PCr values during recovery were fit to an exponential curve, and the rate constant (PCrrate) was calculated. PCrrate was used as an index of oxidative metabolism. Older subjects had lower peak O2 uptake (VO2 peak) values (19.2 +/- 5.6 vs. 49.5 +/- 8.1 ml O2.min-1 x kg-1), CS activities (16 +/- 2.8 vs. 25 +/- 2.6 mmol.kg wet wt-1 x min-1), and PCrrate values (25.3 +/- 8. vs. 37.5 +/- 5.3 mmol PCr.kg wet wt-1.min-1) than young subjects. PCrrate correlated with CS activity, and both PCrrate and CS activity correlated with VO2 peak (P < 0.05). No correlations were found between percent fiber type and PCrrate, CS activity, and VO2 peak. These results support studies that showed decreases in muscle metabolism with age in healthy humans and show a good correlation between in vivo and in vitro measurements of oxidative metabolism.

The response of skeletal muscle to unweighting was studied in six healthy males who were subjected to four weeks of lowerlimb suspension. They performed three bouts of 30 consecutive maximal concentric knee extensions, before unloading and the day after (POST 1), 4 days after (POST 2) and 7 weeks after (REC) resumed weight-bearing. Peak torque of each contraction was recorded and work was calculated as the mean of the average peak torque for the three bouts and fatigability was measured as the decline in average peak torque over bouts. Needle biopsies were obtained from m. vastus lateralis of each limb before and at POST 1. Muscle fibre type composition and area, capillarity and the enzyme activities of citrate synthase (CS) and phosphofructokinase (PFK) were subsequently analysed. Mean average peak torque for the three bouts at POST1, POST2 and REC was reduced (P < 0.05) by 17, 13 and 7%, respectively. Fatigability was greater (P < 0.05) at POST2 than before unloading. Type I, IIA and IIB percentage, Type I and II area and capillaries per fibre of Type I and II did not change (P > 0.05) in response to unloading. The activity of CS, but not PFK, decreased (P < 0.05) after unloading. The weight-bearing limb showed no changes in the variables measured. The results of this study suggest that this human lowerlimb suspension model produces substantial impairments of work and oxidative capacity of skeletal muscle. The performance decrements are most likely induced by lack of weight-bearing.

We examined the effect of long-term intermittent cold exposure on the fiber type composition of the predominantly type I soleus and the predominantly type IIb extensor digitorum longus (EDL) muscles of rats. Cold exposure was accomplished by submerging the rats in shoulder-deep water, maintained at 20 +/- 0.5 degrees C, for 1 h/day, 5 days/wk, for < or = 19 wk. The efficacy of the treatment was tested by subjecting both groups to 20 degrees C water for 45 min while rectal temperature (Tre) and O2 consumption (VO2) were measured. The cold-exposed group displayed a 22% smaller reduction in Tre (P < 0.05) at the end of the exposure and 23% greater VO2 (P < 0.05) during the same period. Fiber type composition was determined using routine histochemical methods for myosin-adenosinetriphosphatase. In the soleus muscle of the cold-exposed rats, the number of type IIa fibers increased 156% (P < 0.05) and the number of type I fibers decreased 24% (P < 0.05). Cold exposure had no significant influence on the fiber type composition of the EDL muscle. Cold exposure resulted in an increase in citrate synthase activity of 20 and 22% in the soleus and EDL muscles, respectively (P < 0.05). The present study demonstrates that intermittent cold exposure induces a type I-to-type IIa transformation in the soleus muscle while having no influence on the EDL muscle.

Eight healthy men performed supine one-legged training on a bicycle ergometer 45 min per leg four times per week for 4 week. The ergometer and lower body were inside a pressure chamber, the opening of which was sealed at the level of the crotch. One leg trained with impeded leg blood flow (I-leg), induced by an increased (50 mmHg) chamber pressure, at the highest tolerable intensity. The contralateral leg trained at the same power under normal pressure (N-leg). Before and after training biopsies were taken from the vastus lateralis of both legs and maximal one-legged exercise tests were executed with both legs. Biopsies were repeated when the subjects had been back to their habitual physical activity for 3 months. Training increased exercise time to exhaustion, but more in the I-leg than in the N-leg. After training, the I-leg had higher activity of citrate synthase (CS), a marker of oxidative capacity, and lower activity of the M-subunit of lactate dehydrogenase isoenzymes. It also had a higher percentage of type-I fibres and a lower percentage of IIB fibres, larger areas of all fibre types and a greater number of capillaries per fibre. It is concluded that ischaemic training changes the muscle metabolic profile in a direction facilitating aerobic metabolism. An altered fibre-type composition may contribute, but is not enough prerequisite for the change.

Insulin- and contraction-stimulated skeletal muscle glucose transport is governed largely by the GLUT-4 isoform of the glucose transporter. Recently, it has been demonstrated that denervated muscle has decreased GLUT-4 protein content, suggesting that regulation of GLUT-4 protein is related to neuromuscular activity. However, until now the effects of the opposite situation, enhanced neuromuscular activity, could only be speculated on from exercise training studies. In the present investigation the effect of chronic low-frequency electrical stimulation (10 Hz, 8 h/day) on GLUT-4 protein content and citrate synthase activity was determined in the predominantly fast-twitch plantaris. Chronic electrical stimulation enhanced GLUT-4 protein content and citrate synthase activity in the muscles stimulated for 10-20 days. Electrical stimulation lasting 30-40 days resulted in no further enhancement of GLUT-4 protein content while citrate synthase activity continued to increase. Further prolongation of electrical stimulation (60-90 days) resulted in a plateauing of citrate synthase activity. The results suggest that increased neuromuscular activity can act independently of systemic changes to increase total GLUT-4 protein content. They also suggest that both GLUT-4 protein content and citrate synthase activity are positively related to increased neuromuscular activity but that their rates of increase differ substantially.

To investigate the effects of exercise training and immobilization on peak O2 uptake (VO2) and effective O2 diffusive conductance (DO2) in skeletal muscle, three groups of purpose-bred hounds [control (C), exercise trained (E), and immobilized (I)] were studied. Group E exercised on a treadmill 1 h/day, 5 days/wk for 8 wk, while groups C and I were cage confined for 8 wk, with group I undergoing left hindlimb immobilization for the last 3 wk. Thereafter, each dog's left gastrocnemius was surgically isolated, pump perfused, and electrically stimulated to elicit peak VO2 in situ at three levels of arterial oxygenation. O2 delivery [(arterial O2 concentration x muscle blood flow)/100 g muscle] was kept constant among the three groups at each level of arterial oxygenation. Compared with group C, peak VO2/100 g muscle was 38, 33, and 19% greater and DO2/100 g muscle was 71, 75, and 68% greater during normoxia, moderate hypoxia, and severe hypoxia, respectively, in group E (P < 0.02), whereas no differences from control were found in group I. We conclude that O2 delivery is not the unique determinant of peak VO2 and that exercise training improves the functional blood-tissue gas exchange properties of the muscle itself. Immobilization sufficient to reduce muscle weight by 31% and citrate synthase activity by 68% has no effect on peak VO2/100 g muscle or DO2/100 g muscle.