Periodic acid Schiff-p phenylenediamine staining of glycogen in chondrocytes

Summary This study reports a method whereby glycogen is identified in the chondrocytes of the secondary center of ossification prior to mineralization. The use of new fuchsin rather than basic fuchsin on one micron Spurr sections of femoral head cartilage fixed with potassium ferrocyanide-reduced osmium produced excellent identification of glycogen and when followed by p phenylenediamine, intensified cellular detail.     

Affinity glycogen synthetase to crosslinked glycogen.

The affinity of yeast glycogen synthetase to glycogen modified by crosslinking has been studied under various experimental conditions. It was found that the higher the degree of crosslinking, the lower the affinity of glycogen synthetase to glycogen. The amount of glycogen synthetase adsorbed from the solution depends on the amount of crosslinked glycogen added and is inversely proportional to the concentration of the soluble glycogen. The stability of the complex formed between yeast glycogen synthetase and the crosslinked glycogen was found to be maximal at neutral pH range. The presence of glucose 6-phosphate, uridine 5′-di-phosphate, and uridine 5′-diphosphate glucose enhanced the stability of the complex.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Characteristic properties of the complex of glycogen synthase with glycogen

The glycogen synthase I--glycogen complex isolated from rabbit skeletal muscles is stable during precipitation with trichloroacetic acid and Sepharose 2B chromatography. The complex catalyzes the synthesis (lengthening) of the alpha-1.4-glucosyl chains when endogenous or exogenous enzyme-free glycogen is used, the initial rates of this synthesis being identical. Preincubation with glycogen does not cause activation of the complex or formation of additional glycogen synthase I--polysaccharide bonds. The complex is characterized by saturation with respect to glycogen; the molar concentration ratios of the non-reducible chain and protein monomer within the complex does not exceed 100. An increase in the length of the synthesized alpha-1.4-glycosyl chains of glycogen results in a decrease of the rate of the glycogen synthase reaction in time.     

Brain glycogen re-awakened

The mammalian brain contains glycogen, which is located predominantly in astrocytes, but its function is unclear. A principal role for brain glycogen as an energy reserve, analogous to its role in the periphery, had been universally dismissed based on its relatively low concentration, an assumption apparently reinforced by the limited duration that the brain can function in the absence of glucose. However, during insulin-induced hypoglycaemia, where brain glucose availability is limited, glycogen content falls first in areas with the highest metabolic rate, suggesting that glycogen provides fuel to support brain function during pathological hypoglycaemia. General anaesthesia results in elevated brain glycogen suggesting quiescent neurones allow glycogen accumulation, and as long ago as the 1950s it was shown that brain glycogen accumulates during sleep, is mobilized upon waking, and that sleep deprivation results in region-specific decreases in brain glycogen, implying a supportive functional role for brain glycogen in the conscious, awake brain. Interest in brain glycogen has recently been re-awakened by the first continuous in vivo measurements using NMR spectroscopy, by the general acceptance of metabolic coupling between glia and neurones involving intercellular transfer of energy substrate, and by studies supporting a prominent physiological role for brain glycogen as a provider of supplemental energy substrate during periods of increased tissue energy demand, when ambient normoglycaemic glucose is unable to meet immediate energy requirements.     

The interaction of muscle glycogen phosphorylase b with glycogen

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.     

Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.     

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.     

Studies of the mechanism of the periodic acid-Schiff histochemical reaction for glycogen using infrared spectroscopy and model chemical compounds.

It has been proposed in the literature that Schiffs reagent reacts with aldehydes to form one of the following types of compounds: alkylsulfonic acids, N-sulfinic acid derivatives, or Schiff bases. Model compounds whose structures are consistent with those proposed in the literature have been synthesized and subjected to infrared analysis. Also, actual products of Schiff reagent reactions with various aldehydes have been isolated and examined using infrared spectroscopy. Comparison of the spectra of the model compounds with those of Schiff-aldehyde reaction products yielded the following conclusions: 1. The reaction of simple organic aldehydes with Schiff's reagent produces an alkylsulfonate-type reaction product. 2. The reaction of periodate-oxidized glycogen with Schiff's reagent probably involves the formation of an alklsulfonate-type compound. 3. The product of the Schiff-aldehyde reaction exists as neither an N-sulfinic acid nor a Schiff base derivative of the fuchsin molecule.     

Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.     

Regulation of glycogen synthesis in human skeletal muscle: does cellular glycogen control glycogen synthase phosphatase activity?

Contrary to the accepted feedback control mechanism of glycogen biosynthesis in skeletal muscle, evidence is presented here leading to the conclusion that glycogen does not control the activity of glycogen synthase phosphatase in intact human skeletal muscle tissue.     

Schistosoma haematobium: histochemistry of glycogen, glycogen phosphorylase a and glycogen branching enzyme in niridazole-treated females.

The body posterior to the ovary of Schistosoma haematobium females was investigated. Glycogen, glycogen phosphorylase a (EC 2.4.1.1) and glycogen branching enzyme (EC 2.4.1.18) activities were detected in the subtegumental muscle system, parenchyma and mature vitelline cells, whereas no activities were detected in the tegument and immature vitelline cells of the parasite. Administration of a single niridazole dose of 250 mg kg-1 to the pouched mouse (Saccostomus camestris) produced the following changes in S. haematobium females: a relatively rapid depletion of glycogen stores due to disruption of the absorptive surface of the parasite, and to an increase in the activity of glycogen phosphorylase a; a reduction in the phosphorylase a to phosphorylase b-conversion capacity of glycogen phosphorylase phosphatase (EC 3.1.3.17); a decrease in glycogen branching enzyme activity; and a relatively rapid degeneration of parasite cells possibly due to their loss of endogenous energy reserves.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

Increased glycogen storage in yeast results in less branched glycogen

Glycogen is a branched polymer of glucose, synthesized as a reserve of both energy and carbon. The branched nature of glycogen is important for its function and polyglucosan bodies, particles that contain a glycogen-like polymer with reduced branching, are a feature of several disease states. The degree of glycogen branching is thought to be governed by the balance between glycogen synthesis and branching activities. However, there have been reports that the intrinsic properties of individual branching enzymes govern the degree of branching. To address the relationship between synthesis and branching more fully, we made use of the yeast Saccharomyces cerevisiae. The glycogen content of yeast cells was manipulated by using different growth conditions or by the introduction of specific mutations. Whenever glycogen storage was elevated, the polysaccharide formed was found to be less branched but normal branching could be restored by overexpression of branching enzyme.     

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.     

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.     

Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively. Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ stimulation of rabbit tibialis anterior muscle, we show GS and GPh intracellular redistribution at the beginning of glycogen resynthesis after contraction-induced glycogen depletion. We identify a new "player," a new intracellular compartment involved in skeletal muscle glycogen metabolism. They are spherical structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism of skeletal muscle glycogen metabolism based on glycogen enzyme intracellular compartmentalization.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

Regulation of glycogen concentration and glycogen synthase activity in skeletal muscle of insulin-resistant rats

The aim of this study was to investigate the effect of insulin resistance on glycogen concentration and glycogen synthase activity in the red and white gastrocnemius muscles and to determine whether the inverse relationship existing between glycogen concentration and enzyme activity is maintained in insulin resistant state. These questions were addressed using 3 models that induce various degrees of insulin resistance: sucrose feeding, dexamethasone administration, and a combination of both treatments (dex+sucrose). Sucrose feeding raised triglyceride levels without affecting plasma glucose or insulin concentrations whereas dexamethasone and dex+sucrose provoked severe hyperinsulinemia, hyperglycemia and hypertriglyceridemia. Sucrose feeding did not alter muscle glycogen concentration but provoked a small reduction in the glycogen synthase activity ratio (-/+ glucose-6-phosphate) in red but not in white gastrocnemius. Dexamethasone administration augmented glycogen concentration and reduced glycogen synthase activity ratio in both muscle fiber types. In contrast, dex+sucrose animals showed decreased muscle glycogen concentration compared to dexamethasone group, leading to levels similar to those of control animals. This was associated with lower glycogen synthase activity compared to control animals leading to levels comparable to those of dexamethasone-treated animals. Thus, in dex+sucrose animals, the inverse relationship observed between glycogen levels and glycogen synthase activity was not maintained, suggesting that factors other than the glycogen concentration modulate the enzyme's activity. In conclusion, while insulin resistance was associated with a reduced glycogen synthase activity ratio, we found no correlation between muscle glycogen concentration and insulin resistance. Furthermore, our results suggest that sucrose treatment may modulate dexamethasone action in skeletal muscle.     

High glycogen levels enhance glycogen breakdown in isolated contracting skeletal muscle

The influence of supranormal muscle glycogen levels on glycogen breakdown in contracting muscle was investigated. Rats either rested or swam for 3 h and subsequently had their isolated hindquarters perfused after 21 h with access to food. Muscle glycogen concentrations were measured before and after 15 min of intermittent electrical muscle stimulation. Before stimulation, glycogen was higher in rats that swam on the preceding day (supercompensated rats) compared with controls. During muscle contractions, glycogen breakdown in fast-twitch red and white fibers was larger in supercompensated hindquarters than in controls, and glycogenolysis correlated significantly with precontraction glycogen concentrations. In slow-twitch fibers, electrical stimulation did not elicit glycogenolysis in either group. Glucose uptake and lactate release were decreased and increased, respectively, in supercompensated hindquarters compared with controls. O2 uptake, release of tyrosine and glycerol, and tension development were similar in the two groups. In conclusion, during muscle contractions, increased muscle glycogen levels lead to increased breakdown of glycogen and release of lactate and decreased uptake of glucose by mechanisms exerted within the muscle cells. Intramuscular lipolysis and net protein breakdown are unaffected. There seems to be no close linkage between needs and mobilization of fuel within the working muscle.