Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.     

Fasting increases plasma membrane fatty acid-binding protein (FABPPM) in red skeletal muscle

The present study was designed to investigate the presence of the fatty acid-binding protein (FABPPM) in the plasma membranes of skeletal muscles with different oxidative capacities for free fatty acid (FFA) oxidation during conditions of normal (fed) or increased (fasted) FFA utilization in the rat. Female Sprague-Dawley rats were either fed or fasted for 12, 24, or 48 h and, plasma membranes (PM) fractions from red and white skeletal muscles were isolated. Short-term fasting significantly decreased body weight by 11% and blood glucose concentration by 42% (6.6 ± 0.2-3.8 ± 0.4 mmol/l) and increased plasma FFA concentration by 5-fold (133 ± 14-793 ± 81 µmol/l). Immunoblotting of PM fractions showed that FABPPM protein content was 83 ± 18% higher in red than in white skeletal muscle and correlated with oxidative capacity as measured by succinate dehydrogenase activity (r = 0.78, p < 0.05). Short-term fasting significantly increased FABPPM protein content by 60 ± 8% in red skeletal muscle but no change was measured in white skeletal muscle. These results show that FABPPM protein content in skeletal muscle is related to oxidative potential and can be increased during a physiological condition known to be associated with an increase in FFA utilization, suggesting that cellular expression of FABPPM may play a role in the regulation of FFA metabolism in skeletal muscle. (Mol Cell Biochem 166: 153-158, 1997)     

The localization of hexokinase isoenzymes in red and white skeletal muscles of the rat

Summary Maximum assayable hexokinase activities vary with the proportion of red, fast-twitch, oxidative-glycolytic and intermediate, slow-twitch, oxidative fibres in different rat skeletal muscles. The major isoenzymic form, type II hexokinase, is present throughout the intermyofibrillar sarcoplasm in all fibres but a proportion of the total activity appears to be weakly associated with mitochondria. Variations in the histochemical staining intensity between fibre types correlate with their mitochondrial content and seem to be due mainly to differences in mitochondrially-associated hexokinase activity. Changes in the strength of this association may be important in controlling increases in glucose metabolism in response to prolonged increased muscular activity while regulation of the equilibrium between free and loosely-bound forms may be an important control feature in all skeletal muscle. Type I hexokinase is a minor isoenzymic component of skeletal muscle and occurs mainly in blood vessels and nerves in the perimysia and endomysia. The majority of this isoenzyme is tightly bound to mitochondria and is not detectable in homogenates prepared in the absence of Triton X-100.     

Metabolic types of muscle in the sheep: I. Myosin ATPase, glycolytic, and mitochondrial enzyme activities

The metabolic characteristics of 12 skeletal muscles of the sheep were studied. Glycolytic activities (hexokinase, glycogen synthetase I and D, phosphorylase a and b, phosphofructokinase) were measured. Myofibrillar ATPase activity was evaluated. Oxygen consumption, respiratory control and carnitine palmityl transferase, isocitrate dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities were measured in isolated mitochondria. Three metabolic types could be distinguished; (1) essentially oxidative slow twitch muscles, typified by the supraspinatus and infraspinatus, having low ATPase activity, (2) fast twitch red muscles, typified by the longissimus dorsi and the semimembranosus, having a higher ATPase activity and both high oxidative and high glycolytic activity, and (3) essentially glycolytic fast twitch muscles, typified by the tensor fascia lata and the semitendinosus, having the highest ATPase activity.     

Mitochondrial gene expression in mammalian striated muscle. Evidence that variation in gene dosage is the major regulatory event

The oxidative capacity of mammalian striated muscles can vary markedly over a nearly 10-fold range, reflecting major differences in the expression of genes that encode enzymes of oxidative metabolism, including genes located exclusively within mitochondrial DNA. To clarify the regulatory events that govern expression of mitochondrial genes in striated muscle, nucleic acid hybridization procedures employing cloned segments of mitochondrial DNA as probes were utilized to determine the concentrations of mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA (a mitochondrial gene product) in rabbit striated muscles of markedly different oxidative capacities. When cardiac muscle and Type I (red, oxidative) skeletal muscle were compared to Type II (white, glycolytic) skeletal muscle, mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA, each increased in direct proportion to increases in oxidative capacity. Furthermore, when the phenotypic characteristics of Type II skeletal muscle were altered by electrical stimulation in vivo, mitochondrial DNA, mitochondrial rRNA, and cytochrome b mRNA also increased proportionately with increases in oxidative capacity. These results indicate that the expression of mitochondrial genes in mammalian striated muscle is proportionate to their copy number, and support the hypothesis that amplification of the mitochondrial genome relative to chromosomal DNA is an important feature underlying enhanced expression of mitochondrial genes in highly oxidative tissues.     

Nature of the changes in the activity of water-soluble enzymes in exposure of muscles to harmful agents. IV. The study of hexokinase extractability from intact and altered muscles

A possibility of hexokinase binding with actomyosin in skeletal muscles of Rana temporaria L., and the effect of thermal alteration (15 min at 36, 37, 38, 40 and 42 degrees C) on the binding were studied. Solutions of KCl (0.075 M and 0.15 M) extract more hexokinase from intact and altered muscles than does an non-electrolyte medium. Hexokinase freely dissolved in hyaloplasm is extracted in non-electrolyte medium. Hexokinase bound with structural components of the muscle cell is extracted upon the increase in ionic force of the extractant. The solubilizing effect of electrolytes on hexokinase is higher in alterated muscles than in the intact muscles indicating the increase in hexokinase binding under thermal alteration. Actomysin isolated from muscles reveals hexokinase activity. In reprecipitated actomyosin, the larger part of its hexokinase remains in actomyosin gel, the level of hexokinase activity not depending on the number of reprecipitation procedures or on the volume of washing solution. Hexokinase in actomyosin gel is less stable to the thermal action than in water supernatant of muscle extract. This may be due to the increase in hexokinase binding with actomiosin whose sorption activity increases under the thermal denaturation.     

Effect of cadmium intoxication on glucose utilization in energy metabolism of muscles

The influence of cadmium intoxication on carbohydrate metabolism in skeletal muscles and liver of the male Wistar rats has been studied. Cadmium was administered as cadmium acetate in a dose of 0.3 mg Cd2+/kg body weight for three months. At the same time the control rats were injected with 0.9% NaCl. The animals were decapitated and samples of their skeletal muscles: the soleus muscle (composed mainly of red slow twitch fibers; ST) the gastrocnemius muscle containing two types of fibers (white fast twitch fibers FTb and red fast twitch fibers, FTa) and the liver were dissected out. In the samples of muscles, liver and serum contents of glycogen, glucose, pyruvate and lactate, as well as activities of hexokinase, pyruvate kinase and lactate dehydrogenase were measured. Intoxication of rats with cadmium for three months resulted in a reduction of glycolytic enzymes in the serum, ST and FTa muscle fibers and in the liver but did not change the activities of glycolytic enzymes in the FTb muscle fibers. The data obtained for the concentrations of glycogen in the liver and skeletal muscles suggest different mechanisms of cadmium influence on glycogen utilization in these organs.     

Incorporation rate of glucose carbon, palmitate carbon and leucine carbon into metabolites in relation to enzyme activities and RNA levels in human skeletal muscles.

The activities (Vmax) of hexokinase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase, phosphofructokinase, lactate dehydrogenase, citrate synthase, cytochrome c oxidase, and 3-OH-acyl-CoA dehydrogenase in human skeletal muscles were compared with the in vitro utilization of glucose and palmitic acid assessed under optimal conditions. Statistically significant correlations between substrate fluxes and enzyme activities were found suggesting that the substrate incorporation rate in vitro in some way reflects the capacity of metabolic pathways. The incorporation rate of leucine into muscle proteins was also statistically significantly correlated to the RNA concentration in the muscle tissue. Glycolytic and glycogenolytic enzymes correlated significantly to each other and correlations were also found between aerobic enzymes supporting the validity of constant proportions between certain key enzymes in human skeletal muscles.     

Metabolic alterations in skeletal muscle of chronically streptozotocin-diabetic rats

This study was accomplished to determine the effects of chronic streptozotocin diabetes and insulin treatment on selected enzymes and substrates used in energy transduction in muscles composed of different muscle fiber types. Triglyceride concentration in all the muscles of diabetic rats was significantly elevated. Glycogen and protein concentrations were unchanged. The enzyme activities of hexokinase and alanine aminotransferase were significantly reduced and 3-hydroxyacyl-CoA dehydrogenase increased in all the muscles. Declines in phosphofructokinase, lactate dehydrogenase, citrate synthase, and succinate dehydrogenase activities were found in the red gastrocnemius and plantaris. Glycerol-3-phosphate dehydrogenase activity was lower than normal in the red gastrocnemius. Insulin treatment to the diabetic rats returned the altered triglyceride content and enzyme activities to normal, with exception of the lower alanine aminotransferase activity in the red gastrocnemius and plantaris. However, this enzyme was significantly ameliorated when compared with the untreated diabetic rats. The findings show that hypoinsulinism has a differential effect on the enzymatic profile of the different skeletal muscle fiber types, with those of the red gastrocnemius being most severely affected. Insulin treatment returned the enzymatic profile of the fiber types in diabetic rats to essentially normal.     

The effect of endurance-training on the maximum activities of hexokinase, 6-phosphofructokinase,citrate synthase,and oxoglutarate dehydrogenase in red and white muscles of the rat

Adult female rats were subjected to an eleven-week endurance-training programme, and, for the first time, the maximum activities of enzymes that can indicate the quantitative capacities of both anaerobic glycolysis and the Krebs cycle in muscle (viz. 6-phosphofructokinase and oxoglutarate dehydrogenase respectively) were measured in heart plus white and fast-oxidative skeletal muscle. No changes were observed in heart muscle. In fast-oxidative skeletal muscle, activities of hexokinase, citrate synthase, and oxoglutarate dehydrogenase were increased by 51, 26, and 33% respectively but there was no effect on 6-phosphofructokinase. These results demonstrate that in red muscle there is no effect of this training programme on the anaerobic capacity but that of the aerobic system is increased by one third. In white skeletal muscle, only the activity of citrate synthase was increased, which indicates that this activity may not provide even qualitative information about changes in capacity of the Krebs cycle.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

Histochemical and biochemical properties of flight muscle fibers in the little brown bat,Myotis lucifugus

Summary The purpose of this investigation was (1) to determine the fiber composition of pectoralis muscle of the little brown bat,Myotis lucifugus; (2) to compare the fiber composition of this muscle with two of the animal's accessory flight muscles; and (3) to study the effect of hibernation on pectoralis muscle fiber composition. Bat skeletal muscle fibers were also compared with those of white laboratory rats (Rattus norvegicus). Bat pectoralis muscles possessed exceptionally high oxidative capacities as indicated by their succinate dehydrogenase activities, but relatively low glycolytic potentials (phosphofructokinase activities). Muscle histochemistry demonstrated that fiber composition of bat pectorlis muscle was homogeneous; all fibers possessed high aerobic and low glycolytic potentials, and high myofibrillar ATPase activities indicating fast contractile properties. In contrast, accessory flight muscles possessed three distinguishable fiber types. During hibernation there was a significant decline in oxidative potential, no change in glycolytic potential, and no alteration in basic fiber composition of bat pectoralis muscle. The findings of this study suggest that pectoralis muscles ofM. lucifugus may approach the ultimate adaptation of a mammalian locomotory muscle for aerobic generation of muscular power.Abbreviations FG fast-twich glycolytic - FOG fast-twitch-oxydative-glycolytic - -GPDH -glycerophosphate dehydrogenase - LDH lactate dehydrogenase - NADH-D reduced nicotinamide adenine dinucleotide diaphorase - PFK phosphofructokinase - SDH succinate dehydrogenase - SO slowtwich-oxidative     

The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.     

C-protein from rabbit soleus (red) muscle.

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.     

A comparative study of glycolysis in red and white muscles of the trout (Salmo gairdneri) and mirror carp (Cyprinus carpio)

The activities of some glycolytic and associated enzymes have been determined in the muscles of trout and carp to investigate the possibility that the discrepancies previously reported between lactate accumulation and anoxic tolerance in these two fish result from underlying differences in glycolytic potential. Steady state concentrations of certain glycolytic intermediates were also determined in freeze-clamped muscles from tankrested fish. The activities of hexokinase, phosphorylase and phosphofructokinase were approximately 2–3 times lower in carp than trout white muscles. Pyruvate kinase and lactate dehydrogenase activities were 5 times lower in carp white muscle. The lower, broader pH optima of lactate dehydrogenase and pyruvate kinase from carp compared to trout muscles is thought to be correlated with the greater anoxic tolerance of the carp. Glycolytic enzyme profiles were markedly different between the red and white muscles of the rainbow trout but broadly similar, with the exception of hexokinase activity, for the corresponding muscles of the carp. The results are discussed in relation to what is known about anaerobiosis in these two species and the comparative physiology of red and white muscles in fish.     

Muscle oxidative capacity during IL-6-dependent cancer cachexia

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.     

Changes in exopeptidase activities in skeletal muscles during disuse

Some aminopeptidase activities, dipeptidase-, tripeptidase-, and carboxypeptidase activities were measured in two different types of skeletal muscle in rabbit soleus muscle as a slow oxidative, and gastrocnemius muscle as a fast glycolytic type after immobilization in full extension with a plaster cast for 1, 2, 4, 7, 14 or 28 days. In correlation to the higher protein turnover in red muscles, the activities except of leucine and alanine aminopeptidase were higher in the normal soleus muscle than in the gastrocnemius muscle. Much higher activities of the tested enzymes were obtained in the immobilized soleus muscle than in the normal one after 2 weeks of immobilization. In the gastrocnemius muscle the tested enzyme activities generally did not change or decrease. The results demonstrate that the peptidases play a role in the process of protein breakdown in normal and disused skeletal muscles.     

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.     

Geographic distribution of xanthine oxidase, free radical scavengers, creatine kinase, and lactate dehydrogenase enzyme systems in rat heart and skeletal muscle.

The geographic distribution of the following enzyme systems is described in the rat heart (left and right ventricles) and in different skeletal muscles (soleus, plantaris, and red and white gastrocnemius): xanthine oxidase and dehydrogenase, creatine kinase isoenzymes, lactate dehydrogenase isoenzymes, and the free radical scavenger enzymes superoxide dismutase, glutathione reductase, and glutathione peroxidase. No substantial difference in enzyme activities was observed between the left and right ventricles. Skeletal muscles showed a clear distinction between enzyme activities depending on their composition of oxidative fibers and glycolytic fibers.