Differential response of rat skeletal muscle glycogen metabolism to testosterone and estradiol.

Although reports on sex steroids have implicated them as promoting protein synthesis and also providing extra strength to the skeletal muscle, it remains unclear whether sex steroids affect glycogen metabolism to provide energy for skeletal muscle functions, since glycogen metabolism is one of the pathways that provides energy for the skeletal muscle contraction and relaxation cycle. The purpose of the current study was to show that testosterone and estradiol act differentially on skeletal muscles from different regions, differentially with reference to glycogen metabolism. To study this hypothesis, healthy mature male Wistar rats (90-120 days of age, weighing about 180-200 g) were castrated (a bilateral orchidectomy was performed to test the significance of skeletal muscle glycogen metabolism in the absence of testosterone). One group of castrated rats was supplemented with testosterone (100 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards). To test whether estradiol has any effect on male skeletal muscle glycogen metabolism 17beta-estradiol (5 microg/100 g body weight, i.m., for 30 days from day 31 postcastration onwards) was administered to orchidectomized rats. To test whether these sex steroids have any differential effect on skeletal muscles from different regions, skeletal muscles from the temporal region (temporalis), muscle of mastication (masseter), forearm muscle (triceps and biceps), thigh muscle (vastus lateralis and gracilis), and calf muscle (gastrocnemius and soleus) were considered. Castration enhanced blood glucose levels and decreased glycogen stores in skeletal muscle from head, jaw, forearm, thigh, and leg regions. This was accompanied by diminished activity of glycogen synthetase and enhanced activity of muscle phosphorylase. Following testosterone supplementation to castrated rats, a normal pattern of all these parameters was maintained. Estradiol administration to castrated rats did not bring about any significant alteration in any of the parameters. The data obtained suggest a stimulatory effect of testosterone on skeletal muscle glycogenesis and an inhibitory effect on glycogenolysis. Estradiol did not play any significant role in the skeletal muscle glycogen metabolism of male rats.     

Glycogen metabolic enzymes in the skeletal muscles of the developing chick embryo

In the skeletal muscles of the chick embryo from the 10th till the 15th day of embryogenesis, phosphorylase (EC. 2.4.1.1) is represented by two isozymes one of which corresponds, by electrophoretic mobility, to the liver phosphorylase and another to phosphorylase of the skeletal muscles of the adult rat. From the 17th day of embryogenesis on only one isozyme of phosphorylase is found in the skeletal muscles which is identical with that of the skeletal muscles of the adult bird. The isozyme spectrum of phosphorylase of the whole 4 days old embryo contains, besides phosphorylase L, a special "embryonic" isozyme which differs from that of the skeletal muscles by immunochemical characteristics and electrophoretic mobility. From the 10th day of embryogenesis till hatching, the activity of phosphorylase of the skeletal muscles increases more than 50 times and that of glycogen synthetase (EC. 2.4.1.11) only 4 times.     

Changes in glycogen metabolism in chick embryo liver in relation to the formation of glycosomes]

In the chick embryo liver the portion of granular glycogen increases from 15 to 90% of the total content during the period from the 8th till the 14th days of developments. The activity of glycogen synthetase (KF 2.4.1.11) localized in the fraction of granular glycogen increases from 40 to 90% of the total activity in the 18 days old embryo. The activity of phosphorylase (KF 2.4.1.1) is detected in the granular glycogen of the liver only on the 12th day of development (10% of the total activity) and increase up to 80% on the 19th day of development. The maximal activation of glycogen synthetase and phosphorylase is noted after the glycosomes of formation in the developing embryoliver. A suggestion is put forward to the effect that the process of glycosome formation is a factor of the control of glycogen synthetase and phosphorylase activity.     

Variation of phosphorylase distribution in skeletal muscles

Summary Glycogen phosphorylase, glycogen alpha-4 UDP-glucosyl transferase, glycogen, and some enzymes were histochemically examined in rat skeletal muscles. Phosphorylase activity was abundantly demonstrated not only in large fibers of the white muscle, but also in small red fibers of soleus muscle and those in the deep fascicles of gastrocunemius and quadriceps femoris muscles. Small fibers with high phosphorylase activity did not always revealed high LDH activity.Native glycogen was abundant mostly in small fibers or in middlesized fibers. Neither glycogen synthetase, nor glycogenolytic enzyme activity was directly proportionate to native glycogen content.On Leave from Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka, Japan.     

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.     

Changes in the characteristics of several carbohydrate metabolism enzymes during differentiation of loach skeletal muscle]

The gradual change of enzymes of glycogen metabolism proceeds during the skeletal muscle differentiation in the loach. The portion of the muscle type phosphorylase in the skeletal muscles of the embryo at the stage of the beginning of movement amounts to 30% and that at the stage of hatching to slightly over 50%. At the stage of yolk resorption, the skeletal muscles contain the muscle type phosphorylase only. At the same time the value of KM(UDPG) for glycogen synthetase gradually increases from 0,1 X 10(-3) up to 0,57 X 10(-3) M. The activity of alpha-glycerophosphate dehydrogenase increases more than 70 times.     

Patterns of amino acid enzyme in domestic fowl breast and leg muscle during development

Activities of alanine and aspartate transaminases, glutamine synthetase, adenylate deaminase, glutamate and xanthine dehydrogenases and lactate dehydrogenase were measured in leg and breast muscles of developing chicks from day 10 in ovo to day 5 of free life, and compared with measurements for adult hens. Xanthine dehydrogenase activity was low in both muscles with adult levels attained on day 15 in ovo. Glutamine synthetase for chicks was maintained higher during development than for adults in both muscles. Minor differences were observed between both muscles in all enzymes tested up to day 18. With low embryonic values and important rises before hatching, the differences were initiated in the posthatching period. Important differences were observed between adult levels of activity. Leg muscle revealed higher enzyme values except for lactate dehydrogenase and indistinguishable levels for adenylate deaminase and xanthine dehydrogenase in both muscles. Alanine, instead of glutamine, is postulated as the main nitrogen transport between muscle and liver in the domestic fowl.     

Regulation of glycogen synthetase. Specificity and stoichiometry of phosphorylation of the skeletal muscle enzyme by cyclic 3':5'-AMP-dependent protein kinase.

Complete conversion of skeletal muscle glycogen synthetase from the I form to the D form requires incorporation of 2 mol of phosphate per enzyme subunit (90,000 g). Incubation of sythetase I with low concentrations of adenosine 3':5'-monophosphate(cAMP)-dependent protein kinase (10 units/ml) and ATP (0.1 to 0.3 mM) plus magnesium acetate (10 mM) results in incorporation within 1/2 hour of 1 mol of phosphate persubunit concomitant with a decrease in the synthetase activity ratio (minus glucose-6-P/plus glucose-6-P) from 0.85 to 0.25. Further incubation for 6 hours does not greatly increase the phosphate content of the synthetase or promote conversion to the D form. This level of phosphorylation is not increased by raising the concentration of protein kinase to 150 units/ml and is not influenced by the presence of glucose-6-P, UDP-glucose, or glycogen. However, at protein kinase concentrations of 10,000 to 30,000 units/ml a second mol of phosphate is incorporated per subunit, and the sythetase activity ratio decreases to 0.05 or less. In addition to the 2 mol of phosphate persubunit which are required for formation of sythetase D, further phosphorylation can be observed which is not associated with changes in synthetase activity. This phosphorylation occurs at a slow rate, is increased by raising the ATP concentration to 2 to 4mM, and is not blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. These data indicate that skeletal muscle glycogen synthetase contains multiple phosphorylation sites only two of which are involved in the synthetase I to D conversion.     

Levodopa with carbidopa diminishes glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in rat skeletal muscle.

We hypothesized that levodopa with carbidopa, a common therapy for patients with Parkinson's disease, might contribute to the high prevalence of insulin resistance reported in patients with Parkinson's disease. We examined the effects of levodopa-carbidopa on glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in skeletal muscle, the predominant insulin-responsive tissue. In isolated muscle, levodopa-carbidopa completely prevented insulin-stimulated glycogen accumulation and glucose transport. The levodopa-carbidopa effects were blocked by propranolol, a beta-adrenergic antagonist. Levodopa-carbidopa also inhibited the insulin-stimulated increase in glycogen synthase activity, whereas propranolol attenuated this effect. Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was reduced by levodopa-carbidopa, although Akt phosphorylation was unaffected by levodopa-carbidopa. A single in vivo dose of levodopa-carbidopa increased skeletal muscle cAMP concentrations, diminished glycogen synthase activity, and reduced tyrosine phosphorylation of IRS-1. A separate set of rats was treated intragastrically twice daily for 4 wk with levodopa-carbidopa. After 4 wk of treatment, oral glucose tolerance was reduced in rats treated with drugs compared with control animals. Muscles from drug-treated rats contained at least 15% less glycogen and approximately 50% lower glycogen synthase activity compared with muscles from control rats. The data demonstrate beta-adrenergic-dependent inhibition of insulin action by levodopa-carbidopa and suggest that unrecognized insulin resistance may exist in chronically treated patients with Parkinson's disease.     

Role of adrenoceptors and cAMP on the catecholamine-induced inhibition of proteolysis in rat skeletal muscle

The role of adrenoceptor subtypes and of cAMP on rat skeletal muscle proteolysis was investigated using a preparation that maintains tissue glycogen stores and metabolic activity for several hours. In both soleus and extensor digitorum longus (EDL) muscles, proteolysis decreased by 15-20% in the presence of equimolar concentrations of epinephrine, isoproterenol, a nonselective beta-agonist, or clenbuterol, a selective beta(2)-agonist. Norepinephrine also reduced proteolysis but less markedly than epinephrine. No change in proteolysis was observed when muscles were incubated with phenylephrine, a nonselective alpha-agonist. The decrease in the rate of protein degradation induced by 10(-4) M epinephrine was prevented by 10(-5) M propranolol, a nonselective beta-antagonist, and by 10(-5) M ICI 118.551, a selective beta(2)-antagonist. The antiproteolytic effect of epinephrine was not inhibited by prazosin or yohimbine (selective alpha(1)-and alpha(2)-antagonists, respectively) or by atenolol, a selective beta(1)-antagonist. Dibutyryl cAMP and isobutylmethylxanthine reduced proteolysis in both soleus and EDL muscles. The data suggest that catecholamines exert an inhibitory control of skeletal muscle proteolysis, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

Reaction of striated fibers of human skeletal muscles to long-term anti-orthostatic hypokinesia

Eight men have been kept under a strict bed rest. The lower end of the bed has been elevated by 6 degrees. During the whole effect of the antiorthostatic hypokinesia (AOH) in biopsy of the skeletal muscle tissue no gross morphological changes and essential changes in relation of the muscle fiber (MF) types have been found. On the 120th day of AOH without application of any physical loading concentration of RNA, protein metabolism, glycogen, activity of the energetic metabolism enzymes, MF size decrease. Both contractile and energetic apparatus suffers essentially. This demonstrates certain atrophic processes in both types of the MF. Physical loading against the background of AOH, on the 120th day, prevents development of the atrophic processes in MF. This is demonstrated as maintenance of certain metabolic level and less pronounced changes of ultrastructure. On the 360th day of the experiment in the group without any loading, an essential atrophy of MF, a noticeable++ decrease in metabolism are observed. In the test group against the background of AOH the changes in the biopsy fibers are less pronounced and not so uniform. Application of physical loadings contributes to the development of certain adaptive reactions; their positive effect to the skeletal muscle fiber morphology depends on the structural background, against which it acts and their intensity. The earlier the load begins to act and the higher its intensity, the more pronounced is the delay in development of profound atrophic changes.     

Quaternary structure of glycogen synthetase I from rabbit skeletal muscles]

Glycogen synthetase I from rabbit skeletal muscles was studied by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. The presence of glycogen in the preparation prevented the destruction of the quaternary structure of the enzyme. In order to separate glycogen synthetase I from glycogen, alpha-amylase from saliva, pig pancrease and bacterial amyloglucosidase were used. The subunit composition of the total preparation and that of the individual glycogen synthetase forms separated ultracentrifugally in the sucrose density gradient, were shown to be identical. The molecular weight of the minimal subunit of glycogen synthetase I from rabbit skeletal muscles was shown to be 36,000. A comparison of the subunit composition of the enzyme preparations stored in the presence and in the absence of phenylmethylsulfanylfluoride did not show that the preparation possesses proteolytic activity.     

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Effect of starvation on branched-chain alpha-keto acid dehydrogenase activity in rat heart and skeletal muscle

The aim of the present study was to investigate changes in the activity of branched-chain alpha-keto acid dehydrogenase (BCKAD) in skeletal muscle and the heart during brief and prolonged starvation. Fed control rats and rats starved for 2, 4 and 6 days were anesthetized with pentobarbital sodium before heart and hindlimb muscles were frozen in situ by liquid nitrogen. Basal (an estimate of in vivo activity) and total (an estimate of enzyme amount) BCKAD activities were determined by measuring the release of 14CO2 from alpha-keto[1-(14)C]isocaproate. The activity state of BCKAD complex was calculated as basal activity in percentages of total activity. Both basal and total activities and the activity state of the BCKAD were lower in skeletal muscles than in the heart. In both tissues, starvation for 2 or 4 days caused a decrease in the basal activity and activity state of BCKAD. On the contrary, in the heart and muscles of animals starved for 6 days a marked increase in basal activity and activity state of BCKAD was observed. The total BCKAD activity was increasing gradually during starvation both in muscles and the heart. The increase was significant in muscles on the 4th and 6th day of starvation. The demonstrated changes in BCKAD activity indicate significant alterations in branched-chain amino acid (BCAA) and protein metabolism during starvation. The decreased BCKAD activity in skeletal muscle and heart observed on the 2nd and 4th day of starvation prevents the loss of essential BCAA and is an important factor involved in protein sparing. The increased activity of BCKAD on the 6th day of starvation indicates activated oxidation of BCAA and accelerated protein breakdown.     

Enzymes of glycogen metabolism in chick embryo liver]

At all stages of ontogenesis glycogen phosphorylase (EC 2.4.1.1) from liver chick embryos in represented by an isoenzyme whose properties are close to those of isoenzyme IL or F. Total enzyme activity (a+b forms) from the 8th day of development up to hatching gradually increases 1.5-fold, a practically complete activation of enzyme being observed by the end of embryogenesis. Phosphorylase b possesses high catalytic activity in the presence of 1 mM AMP and it activated by protamine and 0.2 M Na2SO4. Glycogen synthetase (EC 2.4.1.11) has a constant Km(UDFG) value during ontogenesis. This value is about 5.10(-4) M in the presence of 10 mM glucose-6-phosphate, both for I- and D-forms of enzyme. The total enzyme activity reaches its maximum on the 17th postembryonic day and is decreased more than 6-fold thereafter. In the course of embryogenesis the I/D ratio is increased from 0.2 on the 8th day of development up to 0,45 during extensive accumulation of glycogen and falls down to 0.33 before hatching. Glycogen biosynthesis in embryonic liver is wellcorrelated with the increase in the I/D ratio, i.e. the increase of the active form of enzyme. The proportion of granular glycogen in embryonic liver is increased from 15% up to 90% of total glycogen content between the 8th and 14th days of development. The activity of glycogen synthetase contained in granular glycogen is increased from 40% in the 8-day-old embryos up to 90% in the 18-day-old ones. The activity of phosphorylase is found in granular glycogen only on the 12th day of embryogenesis and reaches its maximum (80% of total enzyme activity) only on the 19th days of development. It is concluded that in the adult chicken liver the embronic enzymes--glycogen phosphorylase and glycogen synthetase--are retained.     

Characteristics of glycogen synthetase I from rabbit skeletal muscles]

Electrophoretic heterogeneity of glycosynthetase I from rabbit skeletal muscles is observed. Multiple glycosynthetase forms are separated in sucrose density gradient, their molecular weights are estimated. The existence of the enzyme as an equilibrium system of oligomeric forms, capable of reversible association-dissociation, is demonstrated. Dissociating effect of ATP, high pH values (11--12) and high ionic strength (2 M KCl) on oligomers of glycogen synthetase I is found to take place. Different activity of oligomers of different association degree is observed.     

Detection and regulation of δ-aminolevulinic acid synthetase activity in rat skeletal muscle

The presence of δ-aminolevulinic acid synthetase (ALAS) in mitochondria obtained from rat skeletal muscles has been observed. Optimal conditions for the meausurement of this activity are described. The activity of skeletal muscle ALAS was investigated under conditions known to affect the activity of this enzyme in other tissues. ALAS activity in skeletal muscle mitochondria was decreased 55% by a 48-h fast. Treatment with dexamethasone did not reverse the effect of starvation on ALAS activity and did not change the activity in the fed controls. ALAS activity was decreased 56% in skeletal muscle mitochondria obtained from rats in which diabetes mellitus had been induced by streptozotocin. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirm our previous suggestion that ALAS activity is regulated in a tissue-specific manner.     

Adrenergic and serotonergic regulation of skeletal muscle metabolism in rat. I. The effects of adrenergic and serotonergic antagonists on the regulation of muscle amino acid release, glycogenolysis, and cyclic nucleotide levels

The biochemical mechanisms of serotonergic and adrenergic action on skeletal muscle cyclic nucleotide, glycogen, and amino acid metabolism have been investigated in intact rat epitrochlaris skeletal muscle preparations. Endogenous catecholamine levels in these preparations were 28.6 +/- 2.1 pg/mg of muscle. Release of these catecholamines by tyramine produced a 25% inhibition of alanine and glutamine release. Pretreatment of animals in vivo with 6-hydroxydopamine depleted catecholamine content by 85%. On incubation, preparations from these pretreated animals showed no effect of tyramine on amino acid metabolism. Serotonin (10(-5) M) and epinephrine (10(-5) M) inhibited alanine and glutamine release equally in preparations from 6-hydroxydopamine-pretreated as compared to control rats. Adrenergic antagonists such as dl-propranolol (10(-8)-10(-6) M), oxprenolol (10(-8)-10(-6) M), and practolol (10(-6)-10(-4) M) blocked equally the inhibition of alanine and glutamine release, prevented the stimulations of muscle cAMP levels, phosphosphorylase a formation, and the depletion of muscle glycogen produced by either epinephrine or serotonin. In contrast, serotonergic antagonists such as methysergide (10(-8)-10(-6) M) and cyproheptadine (10(-8)-10(-6) M) blocked the inhibition of alanine and glutamine release, the stimulations of muscle cAMP levels and phosphorylase a formation, and the decreased muscle glycogen content effected by serotonin but not by epinephrine. Incubation of muscles with both epinephrine and serotonin together produced additive stimulation of muscle cAMP levels, but not of the inhibition of alanine and glutamine release. These data indicate that the action of these agonists on skeletal muscle protein and amino acid, glycogen, and cyclic nucleotide metabolism proceeds directly via separate and discrete serotonergic and adrenergic receptor-adenylyl cyclase mechanisms in skeletal muscle.     

Pentoxifylline inhibits Ca2+-dependent and ATP proteasome-dependent proteolysis in skeletal muscle from acutely diabetic rats

Previous studies from this laboratory have shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cAMP cascade. The present work investigated the systemic effect of pentoxifylline (PTX; cAMP-phosphodiesterase inhibitor) treatment on the rate of overall proteolysis, the activity of proteolytic systems, and the process of protein synthesis in extensor digitorum longus muscles from normal and acutely diabetic rats. The direct in vitro effect of this drug on the rates of muscle protein degradation was also investigated. Muscles from diabetic rats treated with PTX showed an increase (22%) in the cAMP content and reduction in total rates of protein breakdown and in activity of Ca2+-dependent (47%) and ATP proteasome-dependent (23%) proteolytic pathways. The high content of m-calpain observed in muscles from diabetic rats was abolished by PTX treatment. The addition of PTX (10(-3) M) to the incubation medium increased the cAMP content in muscles from normal (22%) and diabetic (51%) rats and induced a reduction in the rates of overall proteolysis that was accompanied by decreased activity of the Ca2+-dependent and ATP proteasome-dependent proteolytic systems, in both groups. The in vitro addition of H-89, an inhibitor of protein kinase A (PKA), completely blocked the effect of PTX on the reduction of proteolysis in muscles from normal and diabetic rats. The present data suggest that PTX exerts a direct inhibitory effect on protein degradative systems in muscles from acutely diabetic rats, probably involving the participation of cAMP intracellular pathways and activation of PKA, independently of tumor necrosis factor-alpha inhibition.     

Antioxidant-induced release of calcium in biological membranes

The effect of synthetic (BHT, 2,2,5,7,8-pentamethyl-6-hydroxychroman) and natural (alpha-tocopherol) antioxidants on Ca++-transporting systems was compared in platelets, brain synaptosomes, and skeletal muscle sarcoplasmic reticulum. It was shown that synthetic antioxidants, in contrast to alpha-tocopherol, induced Ca++-release manifested in platelet aggregation, stimulation of 5-hydroxytryptamine release by synaptosomes, synaptosome depolarization and inhibition of Ca++-transport and Ca++-ATPase activity in the sarcoplasmic reticulum. The disturbances of Ca++-homeostasis induced by synthetic antioxidants are considered as molecular mechanisms of complications encountered upon their application.