Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training.     

Contractile activity modifies Fru-2,6-P(2) metabolism in rabbit fast twitch skeletal muscle.

Modification of muscular contractile patterns by denervation and chronic low frequency stimulation induces structural, physiological, and biochemical alterations in fast twitch skeletal muscles. Fructose 2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. The concentration of Fru-2,6-P(2) depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which catalyzes the synthesis and degradation of this metabolite. This enzyme has several isoforms, the relative abundance of which depends on the tissue metabolic properties. Skeletal muscle expresses two of these isoforms; it mainly contains the muscle isozyme (M-type) and a small amount of the liver isozyme (L-type), whose expression is under hormonal control. Moreover, contractile activity regulates expression of muscular proteins related with glucose metabolism. Fast twitch rabbit skeletal muscle denervation or chronic low frequency stimulation can provide information about the regulation of this enzyme. Our results show an increase in Fru-2,6-P(2) concentration after 2 days of denervation or stimulation. In denervated muscle, this increase is mediated by a rise in liver PFK-2/FBPase-2 isozyme, while in stimulated muscle it is mediated by a rise in muscle PFK-2/FBPase-2 isozyme. In conclusion, our results show that contractile activity could alter the expression of PFK-2/FBPase-2.     

Skeletal Muscle Diseases: Recent Advances and Some Related Basic Problems

Recent advances in the knowledge of the structure and function of voluntary muscle and some of its diseases are reviewed on the basis of a recently published international symposium. Among the subjects discussed are: the specific metabolic role of various muscle cell constituents (e.g. sarcosomes, microsomes, sarcoplasmic reticulum); the relaxing factor system and the dual (tonic and phasic) innervation of muscle fibres; observations on the physiology of muscle training; the morphologic differential diagnosis of degenerative skeletal muscle diseases; the value of serum enzyme determinations in the early detection of muscular dystrophy and in the identification of dystrophic carriers; the classification and diagnosis of muscular hypotonias of infancy; the role of inactivity and the trophic control of the nervous system in the development of neural (or denervation) atrophies; factors influencing regeneration of muscle fibres; the significance, as a research tool, of the identification of hereditary primary muscle disease in laboratory animals.     

Increased muscular dehydroepiandrosterone levels are associated with improved hyperglycemia in obese rats

This study was undertaken to assess the effects of dehydroepiandrosterone (DHEA) administration and exercise training on muscular DHEA and 5α-dihydrotestosterone (DHT) levels and hyperglycemia in diet-induced obese and hyperglycemic rats. After 14 wk of a high-sucrose diet, obese male Wistar rats were assigned randomly to one of three 6-wk regimens: control, DHEA treatment, or exercise training (running at 25 m/min for 1 h, 5 days/wk; n = 10 each group). Results indicate that either 6 wk of DHEA treatment or exercise training significantly attenuated serum insulin and fasting glucose levels compared with the control group. Plasma and muscle concentrations of DHEA and DHT and expression levels of 5α-reductase were significantly higher in the DHEA-treated and exercise-training groups. Moreover, both DHEA administration and exercise training upregulated GLUT4 translocation with concomitant increases in protein kinase B and protein kinase Cζ/λ phosphorylation. Muscle DHEA and DHT concentrations closely correlated with blood glucose levels (DHEA treatment: r = -0.68, P < 0.001; exercise training: r = -0.65, P < 0.001), serum insulin levels, and activation of the GLUT4-regulated signaling pathway. Thus, increased levels of muscle sex steroids may contribute to improved fasting glucose levels via upregulation of GLUT4-regulated signaling in diet-induced obesity and hyperglycemia.     

Effect of reinnervation on collagen synthesis in rat skeletal muscle.

The effect of reinnervation on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) was studied in gastrocnemius, soleus, and tibialis anterior muscles of rat 19, 26, 40, and 61 days after crush denervation of the sciatic nerve. The GGT activity was elevated in denervated gastrocnemius and soleus muscles and the PH activity in gastrocnemius. Muscular Hyp concentration was increased in denervated tibialis anterior muscle. Both the PH and GGT activities and the Hyp concentration returned to the control level during the reinnervation period (19-61 days from the start of denervation). It seems that denervation atrophy of skeletal muscle is associated with an increased rate of muscular collagen biosynthesis and that during reinnervation collagen synthesis rate decreases despite accelerated muscular growth. The results thus suggest that innervation is a powerful suppressive regulator of muscular collagen biosynthesis.     

Respiratory activities of subsarcolemmal and intermyofibrillar mitochondrial populations isolated from denervated and control rat soleus muscles

Ultraturrax and Nagarse released populations of mitochondria isolated from control and day 21 denervated rat soleus muscle were characterized with respect to their oxidative phosphorylation, ADP translocase and ATPase activities. Both Ultraturrax and Nagarse released mitochondrial populations displayed lower capacities for oxidative phosphorylation; lower ADP translocase activities and higher Mg2+ stimulated ATPase activities than their corresponding controls. For both the denervated and control states, the Nagarse-released mitochondrial populations displayed significantly higher respiratory activities than the Ultraturrax released fractions. The significance of these findings is discussed with regard to the process of mitochondrial respiratory control. In addition the role of mitochondrial dysfunction in denervation muscular atrophy is assessed.     

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.     

Oxidative metabolism of rat skeletal muscle mitochondria in adaptation to cold

In control and cold-adapted rats the oxygen consumption was measured polarographically in isolated mitochondria of gastrocnemic and soleus muscles before denervation and 60 min afterwards. Muscle denervation decreased the direct oxidation intensity in both the groups of rats. Unlike, the intensity of phosphorylation in cold-adapted animals increased following the denervation in the soleus muscle, and decreased in the gastrocnemic muscle, whereas no changes were evident in the control rats. It is concluded that the adaptation to cold may augment the dependence of oxidative metabolism in muscle mitochondria on the central nervous control.     

Resveratrol ameliorates metabolic disorders and muscle wasting in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is characterized by dysregulated energy metabolism. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in diabetic animals. However, its overall in vivo effects on energy metabolism and the underlying mechanism require further investigation. In the present study, electrospray ionization-tandem mass spectrometry was employed to characterize the urine and plasma metabolomes of control, streptozotocin-induced DM and RSV-treated DM rats. Using principal component analysis (PCA) and heat map analysis, we discovered significant differences among control and experimental groups. RSV treatment significantly reduced the metabolic abnormalities in DM rats. Compared with the age-matched control rats, the level of carnitine was lower, and the levels of acetylcarnitine and butyrylcarnitine were higher in the urine and plasma of DM rats. RSV treatment ameliorated the deranged carnitine metabolism in DM rats. In addition, RSV treatment attenuated the diabetic ketoacidosis and muscle protein degradation, as evidenced from the attenuation of elevated urinary methyl-histidine and plasma branched-chain amino acids levels in DM rats. The beneficial effects of RSV in DM rats were correlated with activation of hepatic AMP-activated protein kinase and SIRT1 expression, increase of hepatic and muscular mitochondrial biogenesis and inhibition of muscle NF-κB activities. We concluded that RSV possesses multiple beneficial metabolic effects in insulin-deficient DM rats, particularly in improving energy metabolism and reducing protein wasting.     

More than a store: regulatory roles for glycogen in skeletal muscle adaptation to exercise

The glycogen content of muscle determines not only our capacity for exercise but also the signaling events that occur in response to exercise. The result of the shift in signaling is that frequent training in a low-glycogen state results in improved fat oxidation during steady-state submaximal exercise. This review will discuss how the amount or localization of glycogen particles can directly or indirectly result in this differential response to training. The key direct effect discussed is carbohydrate binding, whereas the indirect effects include the metabolic shift toward fat oxidation, the increase in catecholamines, and osmotic stress. Although our understanding of the role of glycogen in response to training has expanded exponentially over the past 5 years, there are still many questions remaining as to how stored carbohydrate affects the muscular adaptation to exercise.     

Glycobiology of neuromuscular disorders

There has been a recent explosion in the identification of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A number of these findings relate to defects in the glycosylation of alpha dystroglycan. Alpha dystroglycan is an essential component of the dystrophin-glycoprotein complex, and aberrant glycosylation of alpha dystroglycan is associated with multiple forms of muscular dystrophy in mice and humans. We review the evidence that defects in dystroglycan glycosylation cause muscular dystrophy. In addition, we review evidence that glycobiology is important in other disorders that affect muscle, including hereditary inclusion body myopathy type II and congenital disorders of glycosylation. Finally, we discuss the long-term potential of glycotherapies for muscle disorders.     

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.     

Effect of diazepam treatment on metabolic indices in trained and untrained rats

Exercise training, like diazepam, is commonly employed as a means of reducing anxiety. Both diazepam and exercise training have been shown to modify carbohydrate and lipid metabolism as well as influence calcium metabolism in skeletal muscle. As receptor binding and thereby efficacy of diazepam has been demonstrated to be modulated by the lipid environment of the receptor, and changes in calcium levels can affect a number of intracellular signalling pathways, we sought to determine if the interaction of both chronic diazepam and exercise training would modify selected metabolic indices in an animal model. For this purpose, muscle and liver glycogen, blood glucose and plasma free fatty acids (FFA) were measured in sedentary, exercise trained and exercise trained, acutely exhausted animals. Alterations in lipid and carbohydrate metabolism were observed in all experimental groups. Diazepam treatment alone exerts metabolic consequences, such as elevated muscle glycogen and plasma FFA and depressed blood glucose levels, which are similar to those observed with exercise training. When animals are acutely exercised to exhaustion, however, differences appear, including a reduced rise in plasma FFA, which suggests that long-term diazepam treatment does influence exercise metabolism, possibly as a result of effects on the sympatho-adrenal system.     

Synthesis of apolipoprotein A1 in skeletal muscles of normal and dystrophic chickens

We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A1 (apo-A1) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following surgical denervation (Shackelford, J. E., and Lebherz, H. G. (1983) J. Biol. Chem. 258, 7175-7180; 14829-14833). In the present work we investigate the effect of avian muscular dystrophy on the synthesis of apo-A1 in chicken muscles. The relative rate of synthesis of apo-A1 and levels of apo-A1 RNA in mature dystrophic breast (fast-twitch) muscle were about 6-fold higher than normal, while synthesis of apo-A1 in breast muscles derived from 2-day-old dystrophic chicks was close to normal. These observations suggest that the elevated apo-A1 synthetic rate in mature dystrophic breast muscle results from a failure of the diseased tissue to "shut down" apo-A1 synthesis to the normal level during postembryonic maturation. Apo-A1 synthesis in the "slow-twitch" lateral adductor muscle of dystrophic chickens was found to be normal. Our work is discussed in terms of the apparent similarities between the effects of surgical denervation and muscular dystrophy on the protein synthetic programs expressed by chicken skeletal muscles.     

Cross‐sectional serum metabolomic study of multiple forms of muscular dystrophy

Muscular dystrophies are characterized by a progressive loss of muscle tissue and/or muscle function. While metabolic alterations have been described in patients’‐derived muscle biopsies, non‐invasive readouts able to describe these alterations are needed in order to objectively monitor muscle condition and response to treatment targeting metabolic abnormalities. We used a metabolomic approach to study metabolites concentration in serum of patients affected by multiple forms of muscular dystrophy such as Duchenne and Becker muscular dystrophies, limb‐girdle muscular dystrophies type 2A and 2B, myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy. We show that 15 metabolites involved in energy production, amino acid metabolism, testosterone metabolism and response to treatment with glucocorticoids were differentially expressed between healthy controls and Duchenne patients. Five metabolites were also able to discriminate other forms of muscular dystrophy. In particular, creatinine and the creatine/creatinine ratio were significantly associated with Duchenne patients performance as assessed by the 6‐minute walk test and north star ambulatory assessment. The obtained results provide evidence that metabolomics analysis of serum samples can provide useful information regarding muscle condition and response to treatment, such as to glucocorticoids treatment.     

Benefits of short-term muscular training in reducing the effects of muscular over-exertion

Muscle soreness, serum creatine kinase (CK), and serum and urinary hydroxyproline levels were examined following muscle over-exertion and resistance training. Seven untrained men performed high intensity leg-extension exercises at a resistance at which they could initially accomplish 90% of their 10 repetitions maximum (10 RM), for 30 minutes to induce muscle over-exertion. In Phase 1 a single bout of muscular over-exertion was followed by a week of rest. Phase 2, the training period, was a repeat of the over-exertion workload of Phase 1 for five consecutive days. Phase 3 was a single bout of over-exertion at a higher 10 RM workload. Normo-responsive and hyper-responsive changes in serum CK were exhibited by different subjects in Phase 1. Muscle soreness was perceived by both groups but to a greater extent in the hyper-responsive group. Serum CK and muscle soreness values were lower during Phase 2 and 3 than in Phase 1. Neither serum nor urinary hydroxyproline levels changed significantly post-exercise. These findings show that a single bout of intense exercise and a brief period of muscular training reduces serum CK and muscle soreness responses following a subsequent single bout of exercise at a higher intensity.     

Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3

Skeletal muscle insulin resistance (IR) is closely linked to hyperglycemia and metabolic disorders. Regular exercise enhances insulin sensitivity in skeletal muscle, but its underlying mechanisms remain unknown. Sestrin3 (SESN3) is a stress-inducible protein that protects against obesity-induced hepatic steatosis and insulin resistance. Regular exercise training is known to increase SESN3 expression in skeletal muscle. The purpose of this study was to explore whether SESN3 mediates the metabolic effects of exercise in the mouse model of high-fat diet (HFD)-induced IR. SESN3^?/? mice exhibited severer body weight gain, ectopic lipid accumulation, and dysregulation of glucose metabolism after long-term HFD feeding compared with the wild-type (WT) mice. Moreover, we found that SESN3 deficiency weakened the effects of exercise on reducing serum insulin levels and improving glucose tolerance in mice. Exercise training increased pAKT-S473 and GLUT4 expression, accompanied by enhanced pmTOR-S2481 (an indicator of mTORC2 activity) in WT quadriceps that were less pronounced in SESN3^?/? mice. SESN3 overexpression in C2C12 myotubes further confirmed that SESN3 played an important role in skeletal muscle glucose metabolism. SESN3 overexpression increased the binding of Rictor to mTOR and pmTOR-S2481 in C2C12 myotubes. Moreover, SESN3 overexpression resulted in an elevation of glucose uptake and a concomitant increase of pAKT-S473 in C2C12 myotubes, whereas these effects were diminished by downregulation of mTORC2 activity. Taken together, SESN3 is a crucial protein in amplifying the beneficial effects of exercise on insulin sensitivity in skeletal muscle and systemic glucose levels. SESN3/mTORC2/AKT pathway mediated the effects of exercise on skeletal muscle insulin sensitivity.     

Skeletal muscle fibers in suspension: a new approach to the study of oxidative and glycolytic metabolism in differentiated muscle

A preparation of suspended fibers from m. flexor digitorum brevis of the rat was characterized with respect to morphological features, and its relevance for the study of muscular metabolism investigated. The activities of oxidative (palmitate and pyruvate oxidation) and glycolytic (lactate formation) pathways were enhanced in myofiber suspensions when compared to intact whole muscle. The rate of glycolysis was stimulated about two-fold by insulin in both the myofiber suspensions and intact muscle. Parameters of oxidative metabolism responded similarly to metabolic effectors in the myofiber suspensions and in intact muscle. It is concluded that the myofiber suspensions have distinct advantages over intact muscle for biochemical and pharmacological studies.     

Carbohydrate metabolism during intense exercise when hyperglycemic

The effects of hyperglycemia on muscle glycogen use and carbohydrate metabolism were evaluated in eight well-trained cyclists (average maximal O2 consumption 4.5 +/- 0.1 l/min) during 2 h of exercise at 73 +/- 2% of maximal O2 consumption. During the control trial (CT), plasma glucose concentration averaged 4.2 +/- 0.2 mM and plasma insulin remained between 6 and 9 microU/ml. During the hyperglycemic trial (HT), 20 g of glucose were infused intravenously after 8 min of exercise, after which a variable-rate infusion of 18% glucose was used to maintain plasma glucose at 10.8 +/- 0.4 mM throughout exercise. Plasma insulin remained low during the 1st h of HT, yet it increased significantly (to 16-24 microU/ml; P less than 0.05) during the 2nd h. The amount of muscle glycogen utilized in the vastus lateralis during exercise was similar during HT and CT (75 +/- 8 and 76 +/- 7 mmol/kg, respectively). As exercise duration increased, carbohydrate oxidation declined during CT but increased during HT. Consequently, after 2 h of exercise, carbohydrate oxidation was 40% higher during HT than during CT (P less than 0.01). The rate of glucose infusion required to maintain hyperglycemia (10 mM) remained very stable at 1.6 +/- 0.1 g/min during the 1st h. However, during the 2nd h of exercise, the rate of glucose infusion increased (P less than 0.01) to 2.6 +/- 0.1 g/min (37 mg.kg body wt-1.min-1) during the final 20 min of exercise. We conclude that hyperglycemia (i.e., 10 mM) in humans does not alter muscle glycogen use during 2 h of intense cycling.(ABSTRACT TRUNCATED AT 250 WORDS)     

PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat

To understand the function of the feeding-stimulatory peptide, galanin (GAL), in eating and body weight regulation, the present experiments tested the effects of both acute and chronic injections of this peptide into the paraventricular nucleus (PVN) of rats. With food absent during the test, acute injection of GAL (300 pmol/0.3 microl) significantly increased phosphofructokinase activity in muscle, suggesting enhanced capacity to metabolize carbohydrate, and reduced circulating glucose levels. It also decreased beta-hydroxyacyl-CoA dehydrogenase activity in muscle, indicating reduced fat oxidation, while increasing circulating non-esterified fatty acids (NEFA) and lipoprotein lipase activity in adipose tissue (aLPL). Chronic PVN injections of GAL (300 pmol/0.3 microl/injection) versus saline over 7-10 days significantly stimulated daily caloric intake and increased the weight of four dissected fat depots by 30-40%. These effects, accompanied by elevated levels of leptin, triglycerides, NEFA and aLPL activity, were evident only in rats on a diet with at least 35% fat. Thus, by favoring carbohydrate over fat metabolism in muscle and reversing hyperglycemia, PVN GAL may have a function in counteracting the metabolic disturbances induced by a high-fat diet. As a consequence of these actions, GAL can promote the partitioning of lipids away from oxidation in muscle towards storage in adipose tissue.