Red Skeletal Muscle Research Articles

Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle.

The effects of exogenous oleate on glucose uptake, lactate production and glycogen concentration in resting and contracting skeletal muscle were studied in the perfused rat hindquarter. In preliminary studies with aged erythrocytes at a haemoglobin concentration of 8g/100ml in the perfusion medium, 1.8mm-oleate had no effect on glucose uptake or lactate production. During these studies it became evident that O(2) delivery was inadequate with aged erythrocytes. Perfusion with rejuvenated human erythrocytes at a haemoglobin concentration of 12g/100ml resulted in a 2-fold higher O(2) uptake at rest and a 4-fold higher O(2) uptake during muscle contraction than was obtained with aged erythrocytes. Rejuvenated erythrocytes were therefore used in subsequent experiments. Glucose uptake and lactate production by the well-oxygenated hindquarter were inhibited by one-third, both at rest and during muscle contraction, when 1.8mm-oleate was added to the perfusion medium. Addition of oleate also significantly protected against glycogen depletion in the fast-twitch red and slow-twitch red types of muscle, but not in white muscle, during sciatic-nerve stimulation. In the absence of added oleate, glucose was confined to the extracellular space in resting muscle. Addition of oleate resulted in intracellular glucose accumulation in red muscle. Contractile activity resulted in accumulation of intracellular glucose in all three muscle types, and this effect was significantly augmented in the red types of muscle by perfusion with oleate. The concentrations of citrate and glucose 6-phosphate were also increased in red muscle perfused with oleate. We conclude that, as in the heart, availability of fatty acids has an inhibitory effect on glucose uptake and glycogen utilization in well-oxygenated red skeletal muscle.

Effect of Endurance Training on the Capacity of Red and White Skeletal Muscle of Mouse to Oxidize Carboxyl‐14C‐labelled Palmitate

Three groups of mice were trained for 1, 4 and 5 months according to different running programs on a motor driven treadmill and the fatty acid oxidation capacity (FAO) and the activities of some enzymes of energy metabolism (cytochrome c oxidase, malate dehydrogenase, triosephosphate dehydrogenase, and lactate dehydrogenase) were determined from m. quadriceps femoris (MQF). Endurance training increased the FAO [5-month training 4 days/week, 30 min/day 22% (p less than 0.05); 1-month training, 7 days/week, 150 min/day 37% (p less than 0.001); 4-month training, 5 days/week, 60 min/day 24% (p less than 0.05)]. The activities of cytochrome c oxidase and malate dehydrogenase increased approx. 30% (p less than 0.001) whereas triosephosphate dehydrogenase and lactate dehydrogenase activities were not prominently influenced by training. The predominantly red part of MQF of untrained animals oxidized palmitate four times faster than the predominantly white part. The activities of cytochrome c oxidase and malate dehydrogenase were two times higher showing pronounced FAO in the red part. Endurance training increased the FAO and activities of oxidative enzymes in the red and white parts and in the whole muscle relatively equally resulting in similar differences between the muscle types after training. The absolute increase in the FAO of the red muscle was, however, manyfold when compared in chemical units to the white muscle.

Purification, Molecular Properties and Ontogeny of Carbonic Anhydrase Isozymes. Evidence for A, B and C Isozymes in Avian and Mammalian Tissues

Carbonic anhydrase A from sheep and chicken red skeletal muscles and carbonic anhydrases B and C from chicken intestine and red cells respectively have been isolated in a high state of purity by affinity chromatography. The subunit and native molecular weights of sheep carbonic anhydrase A determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and Sephadex gel filtration respectively were both approximately 34000. In contrast, carbonic anhydrases A, B and C from chicken exhibited a molecular weight determined by sodium dodecyl sulphate electrophoresis of 30000. The amino acid composition of sheep muscle carbonic anhydrase A was distinct from the B and C isozymes, particularly in basic amino acid content, which was significantly higher for the A isozyme (38 cf. 24–29/mol). The zinc content of sheep carbonic anhydrase A was found to be 0.81 g atom Zn/mol. The specific activities of chicken carbonic anhydrase isozyme differed significantly according to the ratio A:B:C = 1:6:47. In addition, these isozymes differed in their affinities for sulphonamide. Chicken carbonic anhydrase A was approximately 100 times less sensitive to acetazolamide inhibition than the chicken C isozyme. Ontogenetic studies in cats showed carbonic anhydrase C to be the only form of the enzyme present in mid-term fetal animals, with carbonic anhydrase A appearing in muscle in the late stages, and carbonic anhydrase B appearing in caecum within 2 days of birth. The evidence is consistent with previous proposals for a third locus encoding carbonic anhydrase in mammalian and avian species. This isozyme (A) is a monomeric, zinc metalloenzyme, which differs from the extensively studied B and C isozymes of carbonic anhydrase in terms of catalytic efficiency, amino acid composition, molecular weight (in sheep), time of ontogenic appearance, sulphonamide inhibition and tissue distribution.

Metabolism by the rabbit of intravenously administered adenine.

A study was made of the metabolism by the rabbit of adenine administered intravenously at a dose of 35 mg/kg with 100 micronCi of 8-14C-adenine. The infused adenine was removed from the blood in two phases, first by diffusion into the tissues and second by metabolic reactions throughout the body. The adenine equilibrated within a few seconds equally between plasma and red blood cells and between them and kidney, liver, duodenum, lung and heart. Diffusion into skeletal muscle was much slower and into brain slowest. The more gradual disappearance of adenine from blood, and from the rest of the body, with a half-life of about 20 minutes and with complete removal by two hours, was predominantly along three pathways, leading to, after four hours: 1) 74 per cent in adenine nucleotide (mostly AMP, ADP, and (ATP); 2) 12 per cent as unchanged adenine in the urine; and 3) 11 per cent as a mixture in almost equal parts of 8-oxyadenine and 2,8-dioxyadenine in the urine. Conversion of adenine to adenine nucleotide, probably by initial reaction with phosphoribosyl pyrophosphate and adenine-phosphoribosyltransferase, was at widely different rates in the organs with duodenum, kidney, liver, and lung high and heart, red blood cell, skeletal muscle and brain relatively low. Sites of formation of the two oxyadenines, probably by action of xanthine oxidase, were not determined.

Effect of Dietary Protein Restriction or Food Restriction on Oxygen Consumption and Mitochondrial Distribution in Cardiac and Red and White Skeletal Muscle of Rats

The effects of food restriction and dietary protein restriction on the oxygen consumption of three types of muscle in the rat were compared. Control rats were fed a diet containing 27% casein. Two groups of protein-restricted rats were fed isoenergy diets containing 15% or 8% casein. Two groups of food-restricted rats were fed either the 27% or 15% casein diets in amounts equivalent to 35% of the intake of energy. Oxygen consumption was measured in papillary muscle and in fibers from the red (high oxidative) and white (low oxidative) portions of the quadriceps with pyruvate and malate as substrates. Control values (micronlO2/g dry wt/min) were 35.2 + 1.5 (papillary), 20.7+/-1.9 (red), and 7.8+/-0.7 (white). Restriction of dietary protein content failed to alter the oxygen consumption of any of the muscle types. In contrast, involuntary food restriction,combined with a reduction in protein content, caused a reduction in the oxygen consumption of papilary and white skeletal muscle, but the metabolism of red skeletal muscle was not affected. No differences were found in the appearance and distribution of mitochondria in cardiac or red skeletal muscle after examination by electron microscopy, but a depletion of subsarcolemmal mitochondria was evident in white muscle from the food-restricted rats.

Reactive hyperemia in capillaries of red and white skeletal muscle.

Reactive hyperemia was examined in individual capillaries of the red anterior (ALD) and white posterior (PLD) latissimus dorsi muscles of the anesthetized chicken following total blood flow occlusion of 3.5-180 s in length. Capillary red cell velocities were measured by the dual-slit photometric technique. The control capillary velocities in the ALD and PLD were 0.56 +/- .03 (SE) and 0.34 +/- .03 mm/s, respectively. With increasing occlusion lengths, the reactive hyperemia peak velocity, hyperemia duration, and excess flow were found to increase progressively in both muscles suggesting a metabolic origin for the response. The ALD showed a greater peak reactive hyperemia flow than the PLD following occlusion lengths of 7-60 s and a greater excess flow following occlusion lengths of 15 s and longer. However, when these data were normalized to control velocities by calculating the peak-to-control velocity ratio and percent flow debt repayment, no significant differences were found between the two muscles. Furthermore, the reactive hyperemia durations were similar in the two muscles at each occlusion length. These data demonstrate that reactive hyperemia is similar in two muscle types which are known to differ considerably in resting metabolism and anaerobic capacity.

Isolation of nuclei from red and white skeletal muscles of the adult rat

The distinct procedures required for the isolation of nuclei from red and white skeletal muscles of the adult rat in reproducible yields of 15 and 20%, respectively, are described. The purity and preservation of the isolated nuclei were evaluated by phase contrast and electron microscopy, by analyses of RNA and protein content and by RNA polymerase assays.

Affinity-chromatographic isolation and some properties of troponin C from different muscle types.

1. The formation of a complex between troponin I and troponin C that is stable in 6M-urea and dependent on Ca2+ was demonstrated in extracts of vertebrate striated and smooth muscles. 2. A method using troponin I coupled to Sepharose is described for the rapid isolation of troponin C from striated and smooth muscles of vertebrates. 3. Troponin C of rabbit cardiac muscle differs significantly in amino acid composition from troponin C of skeletal muscle. The primary structures of troponin C of red and white skeletal muscle are very similar. 4. The troponin C-like protein isolated from rabbit uterus muscle has a slightly different amino acid composition, but possess many similar properties to the forms of troponin C isolated from other muscle types. 5. The electrophoretic mobilities of the I-troponin C complexes formed from components isolated from different muscle types are determined by the troponin I component.

A comparative electrophoretic analysis of mammalian carbonic anhydrase isozymes: Evidence for a third isozyme in red skeletal muscles

1. 1. Starch gel electrophoretic patterns of carbonic anhydrase (CA) isozymes were examined from tissue extracts of cats, sheep, rabbits and mice. 2. 2. In addition to the widely distributed and extensively studied B and C isozymes, an additional isozyme (called CA-A) was observed. 3. 3. Tissue distribution studies showed the A isozyme to be predominantly localized in red skeletal muscles, although this activity was also observed in white and “mixed” skeletal muscles of the cat, sheep and rabbit, as well as sheep lung and rabbit liver. 4. 4. A, B and C isozymes of carbonic anhydrase from cat, sheep and mice exhibited independent variations in nett surface charge. In terms of decreasing anodal migration, the following results are reported: cat A > C > B; sheep C > B > A; and mouse B > C > A. 5. 5. These results are consistent with the existence of 3 genetic loci encoding carbonic anhydrase in mammalian tissues.

Some biochemical similarities and differences between two jack mackerel species, Trachurus declivis and T. novaezelandiae

1. 1. Six proteins, haemoglobin (two loci), serum esterase (two loci) and lactate dehydrogenuase (two loci) from two species of jack mackerel, Trachurus declivis and T. novaeselandiae, were compared electrophoretically. All six loci were found to be monomorphic. 2. 2. LDH from red and white skeletal muscle tissue were electrophoretically different, but the red form and the white form were found to be identical in both species. 3. 3. Both species share an electrophoretically identical serum esterase, but each species also has an electrophoretically distinct serum esterase. 4. 4. Both species share an electrophoretically identical haemoglobin, but each species also has an electrophoretically distinct haemoglobin. 5. 5. The otolith iron contents of the two species were compared and found to be identical.

Comparative studies on amino and thiol groups in myosins from different sources

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N- ethyl maleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg · ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification thoroughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg · ADP. Alkylation of thiols led to loss of the K +-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca 2+activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3)on alkylation was influenced by the presence of Mg · ADP during the reaction. In the absence of this nucleotide, the Ca 2+-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg · ADP, the Ca 2+-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.

Lipoprotein lipase and uptake of chylomicron triglyceride by skeletal muscle of rats.

Uptake of chylomicron triglyceride and lipoprotein lipase was studied in red and white skeletal muscles, heart, and adipose tissue of rats. Retention of triglyceride fatty acids 10 min after injection was 1.6%/g in heart and adipose tissue, 0.2-0.4%/g in red (soleus and diaphragm) muscles, and 0.1%/g in white (psoas minor) muscles of fed rats. Fasting (24 h) increased retention two- to fourfold in red skeletal muscles and heart, had no effect in white muscles, and decreased retention greater than 75% in adipose tissue. Lipoprotein lipase activity in fed rats was lowest in white muscles and in certain red (posterior belly of digastric and medial head of triceps brachii) musclws, intermediate in soleus and diaphragm muscles and adipose tissue, and highest in heart. Fasting doubled lipoprotein lipase activity in all red skeletal muscles and heart, had no effect in white muscles, and decreased activity 60% in adipose tissue. The findings indicate that triglyceride uptake is related to lipoprotein lipase activity in skeletal muscle and the changes in enzyme activity during fasting divert blood triglyceride to red skeletal muscles.

Calcium-binding proteins in electroplax and skeletal muscle comparison of the parvalbumin and phosphodiesterase activator protein of Electrophorus electricus

A soluble calcium-binding protein has been isolated from the red skeletal muscle of the electric eel ( Electrophorus electricus). The purification procedure involved ammonium sulfate precipitation, gel filtration on Sephadex G-75 in the presence of 45Ca 2+, and chromatography on QAE-Sephadex. This procedure resulted in the isolation of a protein which was homogeneous upon polyacrylamide gel electrophoresis. The calcium-binding protein was found to be a typical parvalbumin by the following criteria: (1) molecular weight of 11 000; (2) pl of 4.7; (3) 1.9 mol Ca 2+ bound per mol protein; K d of approx. 10 −7 M; (4) no detectable phosphorus; (5) amino acid composition included nine residues of phenylalanine, single arginine, and no tyrosine or tryptophan; (6) ε max at 259 nm; (7) 260 nm:280 nm absorbance ratio of 4.78. Only one parvalbumin could be detected in muscle. Immunoprecipitation assay revealed that the parvalbumin was a major soluble component of skeletal muscle (0.10 mg/mg soluble protein), but could not be detected in liver, kidney, brain, spleen, heart or electroplax. Comparison of the parvalbumin with a calcium-binding protein previously isolated from electroplax revealed that the two proteins were different as judged by a variety of chemical criteria. These results suggest that during embryological development of electroplax the parvalbumin is lost and that it is not required for the function of electric tissue.

Immobilized ATP and actin columns as a tool for the characterization and separation of different myosins and active myosin fragments.

A comparative affinity chromatography study using agarose-ATP columns revealed differences between heavy meromyosin subfragment 1 (HMM S-1) preparations obtained from rabbit white skeletal, rabbit red skeletal, bovine cardiac, and chicken gizzard muscle myosins. The characteristic patterns were markedly affected by Ca2+ and Mg2+ ions in a manner typical for each myosin species. Similar differences were also observed on comparing the intact myosins of red and white muscles. It thus became possible to separate, at least partially, mixtures of myosin (or HMM S-1) of different origins. Muscle "acetone-dried powder" was used as chromatographic medium for active myosin fragments in affinity chromatography columns. At low ionic strength the columns bound appreciable amounts of heavy meromyosin (HMM) and of HMM S-1. Binding was reversible and the myosin fragments could be eluted by ATP or magnesium pyrophosphate. The absorption peak of bovine cardiac HMM S-1 was found to be less symmetrical than that of the rabbit white skeletal analog. Chemical modification by trinitrophenylation of white skeletal HMM S-1 was found to affect the affinity of binding and the shape of the absorption peak, thus enabling a partial separation of trinitrophenylated fragment from the unmodified protein using an actin column. The resolving power of ATP columns for the separation of red and white skeletal myosins was increased after trintrophenylation of the proteins.

Subunit composition of fish myofibrils: The light chains of myosin

1. 1. The myosin light chains from red and white skeletal muscles myofibrils, actomyosin and myosin of five fish species have been investigated by polyacrylamide gel electrophoresis. 2. 2. Three light chains were found in myosins of white muscle. Red muscle myosins possess two light chains which do not correspond to any of the light chain components of white muscle myosins. 3. 3. Fish myosins have the same light chains pattern as mammal myosins but show a greater variability of mol. wt.

Isolation and general characterization of myoglobin from the dolphin fish Coryphaena hippurus (L.)

1. 1. Myoglobin was isolated from the red skeletal muscle of the dolphin fish Coryphaena hippurus (L.). The myoglobin was freed from minor components by chromatography on DEAE-Sephadex. 2. 2. The myoglobin migrated as a single component in polyacrylamide gel electrophoresis. Sedimentation equilibrium indicated a homogeneous preparation. The molecular weight was determined by sedimentation equilibrium and by detergent-gel electrophoresis. 3. 3. Absorption maxima and molar absorption coefficients were determined for the ferrimyoglobin. The pK′ for the acid dissociation of the haem-bound water molecule in the ferryimyoglobin was shown to be 8·89. 4. 4. The amino acid composition was determined. The myoglobin showed a low histidine and a high aspartic acid content.

Role of myoglobin in the oxygen supply to red skeletal muscle

The contribution of nyoglobin to the oxygen uptake of red skeletal muscle was estimated from the difference in oxygen uptake with and without functional myoglobin. The oxygen uptake of bundles (25 mm long, 0.5 mm mean diameter) of muscle fibers teased from pigeon breast muscle was measured in families of steady states of oxygen pressure from 0 to 250 mm Hg. The oxygen-binding function of myoglobin, in situ in muscle fiber bundles, was abolished by treatment with nitrite of hydroxylamine, which convert oxymyoglobin in situ to high spin ferric myoglobin, or with phenylhydrazine, which converts oxymyoglobin to denatured products, or with 2-hydroxyethylhydrazine, which appears to remove myoglobin from the muslce. The oxygen uptake was again measured. At higher oxygen pressure, where oxygen availability does not limit the respiration of the fiber bundles, oxygen uptake is not affected by any of the four reagents, which is evidence that mitochondrial oxygen uptake is not impaired. At lower oxygen pressure, where oxygen uptake is one-half maximal, the steady state oxygen consumption is roughly halved by abolishing functional myoglobin. Under the steady state conditions studied, the storage function of myoglobin, being static, vanishes and the transport function stands revealed. We conclude from these experiments that myoglobin may transport a significant fraction of the oxygen consumed by muscle mitochondria.

Isozymes of AMP deaminase in red and white skeletal muscles

Isozymes of AMP deaminase in red and white skeletal muscles

Phosphorylation of the light-chain components of myosin from cardiac and red skeletal muscles.

1. The light-chain components of myosin from cardiac muscle (19000 and 27000 daltons) and of rabbit soleus and crureus muscles (19000, 27000 and 29000 daltons) were characterized. 2. The 19000-dalton components in carciac- and red-skeletal-muscle myosins were spontaneously modified to a component of slightly higher net negative charge. 3. The 19000-dalton component in cardiac and red skeletal muscles and their modified forms were phosphorylated by myosin light-chain kinase. 4. Evidence was obtained for the presence of myosin light-chain kinase in cardiac and red skeletal muscles. 5. Myosin light-chain kinase catalysed the phosphorylation of the whole light-chain fraction from white and red skeletal muscle at similar rates. The light-chain fraction of cardiac-muscle myosin was phosphorylated at a significantly lower rate. 6. The light-chain components of cardiac-muscle myosin and their phosphorylated froms were separated by ion-exchange chromatography and their amino acid compositions determined.

Ontogeny and regulation of fructose diphosphate aldolase isoenzymes in "red" and "white" skeletal muscles of the chick.

The quantitative and qualitative changes in fructose-P2 aldolase isoenzyme concentrations during development of "red" (leg) and "white" (breast) skeletal muscles of the chick were investigated. (a) The aldolase C to A subunit transition associated with muscle development is accompanied by large increases in aldolase activity (units/g, wet weight) and in specific catalytic activity (units/mg of protein). The accumulations in both muscle types follow pseudo-first order kinetics with doubling times of 2 to 3 days. The steady state level of aldolase activity in breast muscle (about 150 units/g) is approximately 4-fold higher than that in leg muscle (about 40 units/g). In contrast to leg muscle, the major increase in aldolase activity in breast muscle occurs during postembryonic development. (b) Immunotitration studies demonstrated a direct correlation between increases in enzyme activity and aldolase A subunits during postembryonic muscle development. It was calculated that under steady state conditions, aldolase A4 comprises about 1 percent and 0.26 percent, respectively, of the total wet weight of breast and leg muscle. (c) regulation at the level of protein synthesis in effecting the postembryonic accumulation of aldolase A4 in the muscle types was investigated in short term amino acid incorporation experiments. After a 1-hour pulse with [3H]leucine, aldolase from breast and leg muscle was isolated in a single step by affinity chromatography on phosphocellulose. Incorporation of tritum into aldolase A4 and into soluble or total protein was compared. Between 4 and 38 days after hatching, the rate of aldolase synthesis relative to the synthesis of soluble muscle protein increased about 7- and 3-fold, respectively, in breast and leg muscle. Relative to total protein, incorporation of [3H]leucine into A4 increased about 3-fold in breast muscle, and decreased slightly in leg muscle between 5 and 25 days after hatching. By 3 weeks after hatching, incorporation of [3H]leucine into aldolase A4 relative to incorporation into total protein was about 6-fold higher in breast muscle than it was in leg muscle. The present work, as well as other recent studies, are discussed in relation to the mechanism involved in controlling tissue-specific and stage-specific levels of aldolase isoenzymes in animal cells.