Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.     

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.     

Glyconeogenic and glycogenic enzymes in chronically active and normal skeletal muscle

The chronically active (pseudomyotonic) gastrocnemius muscle in the C57B16J dy2J/dy2J mouse contains both elevated lactate and glycogen as well as fibers that have high amounts of glycogen and enhanced glyconeogenic activity. In the present study we analyze the activities of some key glyconeogenic enzymes to assess the causes of elevated muscle glycogen and to determine the pathway for glycogen synthesis from lactate. Glycogen synthase, malate dehydrogenase, phosphoenolpyruvate carboxykinase, and malic enzyme were all elevated in homogenates of the chronically active muscle. Activities of glycogen phosphorylase and fructose 1,6-bisphosphatase were decreased in whole muscle homogenates. Histochemistry demonstrated that the high-glycogen fibers were typically fast-twitch glycolytic fibers that had high glycogen synthase, glycogen phosphorylase, and malic enzyme activities. Malate dehydrogenase activity followed succinate dehydrogenase activity and did not correlate to high-glycogen fibers. Thus the high-glycogen fibers have an elevated enzymatic capacity for glycogen synthesis from lactate, and the pathway may involve use of the pyruvate kinase bypass enzymes.     

Fiber-specific responses of muscle glycogen repletion in fasted rats physically active during recovery from high-intensity physical exertion

Mild physical activity performed immediately after a bout of intense exercise in fasting humans results in net glycogen breakdown in their slow oxidative (SO) muscle fibers and glycogen repletion in their fast twitch (FT) fibers. Because several animal species carry a low proportion of SO fibers, it is unclear whether they can also replenish glycogen in their FT fibers under these conditions. Given that most skeletal muscles in rats are poor in SO fibers (<5%), this issue was examined using groups of 24-h fasted Wistar rats (n=10) that swam for 3 min at high intensity with a 10% weight followed by either a 60-min rest (passive recovery, PR) or a 30-min swim with a 0.5% weight (active recovery, AR) preceding a 30-min rest. The 3-min sprint caused 61-79% glycogen fall across the muscles examined, but not in the soleus (SOL). Glycogen repletion during AR without food was similar to PR in the white gastrocnemius (WG), where glycogen increased by 71%, and less than PR in both the red and mixed gastrocnemius (RG, MG). Glycogen fell by 26% during AR in the SOL. Following AR, glycogen increased by 36%, 87%, and 37% in the SOL, RG, and MG, respectively, and this was accompanied by the sustained activation of glycogen synthase and inhibition of glycogen phosphorylase in the RG and MG. These results suggest that mammals with a low proportion of SO fibers can also replenish the glycogen stores of their FT fibers under extreme conditions combining physical activity and fasting.     

Insulin stimulates dephosphorylation of phosphorylase in rat epitrochlearis muscles

We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Pi to label cellular phosphoproteins, before being treated with hormones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control and insulin on phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the beta subunit of phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase alpha subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in phosphorylase and phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.     

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.     

Glucagon control of glycogenolysis in catfish tissues

1. In catfish (Ictalurus melas) after glucagon treatment blood glucose increased until 150 min, then it gradually decreased towards control values at the 5th hr. 2. In glucagon treated fish, liver glycogen levels were significantly lower then in controls 30 min after hormone administration; thereafter, liver glycogen levels returned rapidly to initial values. Glucagon did not induce any significant effect on the glycogen content in white and red muscles. 3. In liver slices, the addition of glucagon to the incubation medium significantly enhanced the glycogen phosphorylase activity and decreased the level of glycogen. Both phosphorylase activity and glycogen content of white and red muscle slices were practically unaffected by glucagon.     

Glycogen supercompensation in rat soleus muscle during recovery from nonweight bearing

The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.     

Proteomic analysis of slow- and fast-twitch skeletal muscles

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.     

Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism.

The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.     

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.     

Metabolic differentiation of homologous leg muscles of two aquatic birds at the level of enzymatic organization

The quantitative determination of succinic dehydrogenase (SDH), hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (G-3-PDH) and lactate dehydrogenase (LDH) was carried out in the homologous leg muscles of two aquatic Birds. It appears that the leg muscle fibres of the coot, a surface swimmer are more oxidative in nature and appear to utilize glucose as source of energy. The leg muscles of the dabchick, a diving Bird, on the other hand, seem to depend on glycogen as source of energy. The relative activity levels of HK, phosphorylase and PFK support the accepted r?le of glycogen as primary substrate of carbohydrate catabolism in the leg muscles. The ratio of G-3-PDH/LDH in the leg muscles revealed that glycerol 3-phosphate cycle appears to be insufficient to account for the major part of NADH oxidation. However, the LDH activity is quite high in all the muscles. These results led us to believe that glycerol 3-phosphate cycle may function during rest, when the rate of glycolysis will be low.     

Novel findings on ultrastructural protection of skeletal muscle fibers during hibernation of Daurian ground squirrels: Mitochondria,nuclei, cytoskeleton,glycogen

We examined ultrastructure protective phenomena and mechanisms of slow and fast muscles in hibernating Daurian ground squirrels (Spermophilus dauricus). Some degenerative changes such as slightly decreased sarcomere length and vacuolization occurred in hibernation, but periaxonal capsular borders in intrafusal fibers remained distinct and the arrangement of extrafusal fibers and Z-lines unscathed. In soleus samples, the number of glycogenosomes more than tripled during hibernation. The expression of phosphorylated glycogen synthase remained unaltered while that of glycogen phosphorylase decreased during hibernation. The number of extensor digitorum longus glycogenosomes decreased and the expression of phosphorylated glycogen synthase decreased, while glycogen phosphorylase expression remained unaltered. The nuclei number remained unchanged. Kinesin and desmin, preventors of nuclear loss and damage, were maintained or just slightly reduced in hibernation. The single-fiber mitochondrial concentration and sub-sarcolemmal mitochondrial number increased in both muscle types. The expression of vimentin, which anchors mitochondria and maintains Z-line integrity, was increased during and after hibernation. Also, dynamin-related protein 1, mitochondrial fission factor, and adenosine triphosphate synthase were elevated in both muscle types. These findings confirm a remarkable ultrastructure preservation and show an unexpected increase in mitochondrial capacity in hibernating squirrels.     

Comparative histochemical study of prosimian primate hindlimb muscles. I. Muscle fiber types

The profiles of fiber types in hindlimb muscles from the tree shrew (Tupaia glis), lesser bushbaby (Galago senegalensis), and the slow loris (Nycticebus coucang) were determined using histochemical techniques. Fibers were classified as fast-twitch oxidative-glycolytic (FOG), fast-twitch glycolytic (FG), slow-twitch oxidative (SO), or fast-twitch oxidative (FO), according to reactions for alkaline-stable ATPase, acid-stable ATPase, alpha-glucan phosphorylase, reduced nicotinamide adenine dinucleotide diaphorase, succinate dehydrogenase, mitochondrial alpha-glycerophosphate dehydrogenase (MaGPDH), and beta-hydroxybutyric dehydrogenase, as well as glycogen staining by the periodic acid-Schiff technique. Prolonged dissection of numerous muscles was carried out on hindlimbs submersed in cold Tyrode's solution; such treatment had no qualitative effect on enzyme staining reactions, but it is not a suitable procedure if one wishes to stain for glycogen. Fast-twitch oxidative (FO) fibers are alkaline-stable ATPase-positive and possess low MalphaGPDH enzyme activity. These fibers have not been reported previously in any hindlimb muscles. No muscles of any species studies were homogeneous with respect to fiber type. Slow loris muscles lacked FG fibers. The majority of the muscles of the slow loris contained numerous SO fibers. The relationship between enzyme activities and locomotor pattern is discussed.     

Peculiarities of Activation of Glycogenolysis Enzymes in Skeletal Muscles of the Trout Salmo trutta morpha Fario

In skeletal muscles of the trout, a fish that intensively swims and is capable for sharp sprinting movements, an active form of ATP: phosphorylase b phosphotransferase (EC 2.7.1.38, glycogen phosphorylase kinase; GPK) and partially active 1,4-D-glucan:orthophosphate glucosyltransferase (EC 2.4.1.1, glycogen phosphorylase; GP) are revealed in the state of a relative rest. The isolated GP ab has a higher affinity to substrates (glucose-1-phosphate and glycogen) than GP b and is able to split glycogen without pre-activation with AMP or GPK. The presence of the activated forms of GPK and GP in resting muscles of the trout provides an opportunity for the very fast Ca²⁺-activation of glycogenolysis, coupled with activation of muscle contraction. This seems to be a biochemical mechanism of adaptation for the energy supply of intense muscle activity in this fish species inhabiting rapid cataracted rivers.     

Timing and sequence of the events in the development of extraocular muscles in staged human embryos: ultrastructural and histochemical study.

The ultrastructure and the appearance of glycogen were studied in the extraocular muscles of 14 externally normal human embryos (Carnegie stages 13-21). At stage 16, myofibrils with an immature Z line and glycogen granules appeared in the cytoplasm of the myoblast. The myoblasts came into cluster at stage 18, and fusion between the myotubes was observed at stage 20. At this stage, an M line appeared in the myofibrils. At stage 21, an A band with a Z line and an H band with an M line were observed, the sarcoplasmic reticulum appeared in the cytoplasm of the muscle fibers and glycogen increased in volume in the cytoplasm. In the previous study, we showed that the muscle-specific isoenzymes, such as creatine kinase, beta-enolase and glycogen phosphorylase, appeared from stage 18 to 20 in the extraocular muscles. The previous findings and the present results suggest that the fusion of the muscle cells occurs in the period when some molecular markers of muscle differentiation are expressed in vivo.     

Relationships between mitochondrial content of muscle fibres and patterns of glyogendepletion postmortem.

Sternomandibularis muscles were removed from slaughtered adult cattle immediately after exsanguination. On the basis of the density of diformazan granules deposited by a reaction for NAD tetrazolium reductase, approximately equal numbers of muscle fibres with high and low mitochondrial content were identified in serial frozen sections. In samples taken immediately after exanguination both types of muscle fibres exhibited glycogen phosphorylase activity and were stained equally by the periodic acid-Schiff (PAS) reaction for glycogen. In unstimulated muscle samples 1 hr postmortem, no loss of PAS staining was detected. In electrically stimulated samples 1 hr postmortem, large numbers of muscle fibres with a low mitochondrial content but only some muscle fibres with a high mitochondrial content became PAS-negative. Stimulation-induced glycogen depletion was completely prevented by the interfaicular injection of magnesium sulphate solution. In unstimulated samples between 5 and 24 hr postmortem, some muscle fibres with a high mitochondrial content but only a few muscle fibres with a low mitochondrial content became PAS-negative.     

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.     

Methadone inhibition of glycogen synthase and phosphorylase in rat skeletal muscle

The activities of glycogen synthase (I and total) and phosphorylase ($\text{a}$ and total) in crude extracts of isolated extensor digitorum longus and soleus muscles of the rat incubated $\text{in vitro}$ in the absence or presence of methadone were very low. Addition of glycogen during homogenization increased the activities of both enzymes in control muscles. Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles. The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen. It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone.