Activation of glycogen phosphorylase by electrical stimulation of isolated fast-twitch and slow-twitch muscles from rat

The influence of muscle contraction, induced by electrical stimulation, on the activity of glycogen phosphorylase, the contents of high-energy phosphates, hexose-monophosphates and lactate have been studied in isolated extensor digitorum longus (EDL) and soleus muscles from rats. The activity of phosphorylase a + b was about nine times higher in fast twitch muscles (EDL) than in slow-twitch soleus and remained unchanged during the stimulation. A pronounced increase of phosphorylase a occurred during the stimulation in EDL muscle. Stimulation with a frequency of 50 Hz for 10 s and 2 Hz for 90 s resulted in a 44-fold and five-fold increase in phosphorylase a, respectively. In contrast, stimulation of soleus muscle resulted in only a minor increase of phosphorylase a. The rate of glycogenolysis increased in both muscles during the stimulation but the increase was four to five times higher in the EDL than in soleus muscle. The content of phosphocreatine (PCr) before stimulation was much higher in EDL than in soleus but similar after the stimulation. This resulted in a three- to four-fold higher release of inorganic phosphate (Pi) in EDL than in soleus during contraction. Pi has previously been shown to be present in a limiting amount for the activity of phosphorylase and the increase during contraction is of importance for increasing the glycogenolytic rate. It is concluded that the higher glycogenolytic capacity in fast-twitch muscles compared to slow-twitch muscles is due to: (1) higher content of phosphorylase a + b, (2) higher degree of transformation of the enzyme into the a form during contraction, and (3) higher content of PCr, which liberates a large amount of Pi during contraction.     

Effects of age on glycogen synthase and phosphorylase activities in rat liver

Six exopeptidases present in human diploid fibroblasts were identified by separation on polyacrylamide gel electrophoresis and their activity profiles against 17 dipeptides, two tripeptides and L-leucine-p-nitroanilide determined. No differences in relative activity or in the electrophoretic patterns of any of the six exopeptidases were detected with ageing. Aminoacylarylamidase activity assayed spectrophotometrically showed significantly increased activity in the middle age-group cells as opposed to the enzyme isolated from young and old cells. Heat-inactivation studies using the same substrate suggested the possibility of an increased proportion of heat-labile enzyme in the old cells but interpretation of the data was difficult because of the complex nature of the inactivation curves obtained. Overall, the results tended to refute the hypothesis that age-related changes in the free amino acid pool of human diploid fibroblasts were associated with significant alterations in the activities of cellular exopeptidases.     

Insulin-independent glycogen supercompensation in isolated mouse skeletal muscle: role of phosphorylase inactivation

Glycogen supercompensation (increase in muscle glycogen content above basal) is an established phenomenon induced by unknown mechanisms. It consists of both insulin-dependent and -independent components. Here, we investigate insulin-independent glycogen supercompensation in isolated, intact extensor digitorum longus muscles from mice. Muscles were stimulated electrically, incubated in vitro with 5.5 mM glucose for up to 16 h and then analysed for glycogen, glucose uptake and enzyme activities. Basal glycogen was 84±6 µmol glucosyl units/g dry muscle and was depleted by 80% after 10 min contraction. Glycogen increased after contraction, reaching a peak value of 113±9 µmol glucosyl units/g dry muscle (P<0.05 vs. basal) by 6 h, and returned to basal values by 16 h (84±8). Maximal activities of glycogen synthase, phosphorylase and -glucosidase were not significantly altered by contraction or during the 6-h recovery period. Glycogen synthase fractional activity (0.17/7.2 mM glucose-6-P; inversely related to phosphorylation state of the enzyme) was increased about twofold early after contraction but then decreased and was slightly lower than baseline during the period of supercompensation (4–6 h). Phosphorylase fractional activity (±adenosine monophosphate; directly related to phosphorylation state of the enzyme) decreased to 60% of basal after contraction and decreased further during the initial 4 h of recovery to 40% of basal (P<0.01 vs. basal). After 4 h recovery, glucose uptake was slightly (50%) higher in the stimulated than in the non-stimulated muscle (P<0.01). Thus, insulin-independent glycogen supercompensation involves inactivation of phosphorylase and hence an inhibition of glycogen breakdown.     

Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles

This study investigated the effect of adrenaline infusion on the cAMP content, glycogen phosphorylase activity and the rate of glycogen breakdown in rat extensor digitorum longus (EDL) and soleus muscles. Adrenaline was constantly infused in a dose of 0.15 micrograms kg-1 body wt min-1. The cAMP content increased approximately 2.8-fold in both muscles after 2 min of infusion. Phosphorylase a + b activity was six times higher in fast-twitch muscle (EDL) than in slow-twitch (soleus) and remained unchanged during the infusion. Phosphorylase a activity increased by 8.4-fold in EDL and 2.4-fold in soleus muscles during the infusion period. Glycogen content decreased in EDL muscle by 10% whereas no change was observed in soleus. It is concluded that beta-adrenergic stimulation by adrenaline results in a similar cAMP increase in both muscles. The low rate of glycogen breakdown in EDL and the unchanged content of glycogen in soleus muscle suggest that cAMP mediated transformation of phosphorylase b to a in itself is not adequate for a rapid glycogenolysis in muscle.     

Modulation of glycogen metabolism of rat skeletal muscles by endurance training and testosterone treatment

The effects of training and/or testosterone treatment on glycogen content and the activities of glycogen synthase, glycogen phosphorylase, and fructose-6-phosphate kinase were studied in extensor digitorum longus (EDL) and soleus muscles of intact adult female rats. One group of rats remained sedentary, whereas another group was trained for 7 weeks. Thereafter, both the sedentary and trained rats were subdivided into two control and four testosterone-treated subgroups. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. On the final day of the experiment rats from one trained control and one trained testosterone-treated subgroup ran for 60 min submaximally. Upon testosterone treatment of sedentary rats the glycogen concentration was not changed. However, in the soleus, but not in the EDL, the glycogen content was increased by training (P<0.05) which could, at least partly, be explained by a decrease in activity of active glycogen phosphorylase (P < 0.05). In the EDL of trained rats testosterone treatment increased glycogen content significantly by both an increase in activity of active glycogen synthase and a decrease in activity of active glycogen phosphorylase (P<0.05). In the EDL and soleus of testosterone-treated animals from the exercised subgroup a significant sparing of glycogen was observed, which could be explained by an increase in activity of active glycogen synthase and, in the soleus, could also be explained by a concerted decrease in active glycogen phosphorylase (P<0.05). In the two muscles studied, we also found that testosterone treatment in trained animals shifted the fibre type distribution towards more oxidative fibres in both types of muscle in comparison with the control animals. We conclude that testosterone, at a pharmacological dose, potentiates the training-induced increase in glycogen content of skeletal muscle and induces a glycogen-sparing effect after submaximal exercise.An Established Investigator of the Netherlands Heart Foundation     

Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose

Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120 min after per os administration of glucose or fructose to fasted rats with CCl₄-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20 min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60 min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.     

Effects of endurance training on alkaline protease activities in rat skeletal muscles

This study aimed at comparing the effects of running and swimming training protocols and the termination of training on the activities of two proteases with alkaline pH-optima (alkaline protease and myofibrillar protease) in the tibialis anterior, soleus, and gastrocnemius muscles of male rats. The training on treadmill decreased the activities of alkaline and myofibrillar proteases by approx. 10-20% in the muscles studied. The activities of both proteases were unchanged in swimming-trained rats. Two weeks after the termination of running training the activity of alkaline protease was increased in gastrocnemius muscle but not in the other muscles. Swimming training increased the activity of citrate synthase in all muscles studied but training by running only in the soleus muscle. The running protocol increased the activity of beta-glucuronidase in the tibialis anterior muscle and decreased the activity in the gastrocnemius muscle. The swimming program did not affect beta-glucuronidase activities. These results show diverse effects of running and swimming training on alkaline proteolytic activities as well as on mitochondrial and lysosomal marker enzymes.     

Glycogen synthetase and phosphorylase activity in slow and fast twitch skeletal muscle fibres in man.

Glycogen synthetase (I + D) and phosphorylase (à + b) activity was determined in human skeletal muscle biopsies with different muscle fibre composition and in dissected an pooled batches of the two main muscle fibre types: slow twitch (ST) and fast twitch (FT), respectively. Glycogen synthetase (I + D) activity remained unchanged as the per cent of FT fibres increased, but phosphoyrlase (à + b) activity was found to increase. A similar activity pattern was found in dissected and FT fibres as compared to ST fibres, i.e. the same glycogen synthetase activity but heightened phosphoyrlase activity.     

Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation

AIM: Effects of in vivo adrenaline infusion on subsequent insulin-stimulated glucose uptake and glycogen synthase activation was investigated in slow-twitch (soleus) and fast-twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. METHODS: Male Wistar rats received adrenaline from osmotic mini pumps ( approximately 150 microg kg(-1) h(-1)) for 1 or 12 days before muscles were removed for in vitro studies. RESULTS: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin-stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin-stimulated PKB ser(473) phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin-stimulated PKB phosphorylation was normal in both muscles. CONCLUSION: Insulin-stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin-stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.     

Age-related change in adrenergic regulation of glycogen phosphorylase in rat hepatocytes

The effects of development on adrenergic regulation of glycogenolysis were studied in rat liver. In isolated hepatocytes, the activation of glycogen phosphorylase by alpha-adrenergic stimulation decreased moderately with advancing age. However, activation by beta-adrenergic receptors more markedly declined and almost disappeared in isolated hepatocytes from 6-month-old rats. Furthermore, the ability to glucagon to stimulate phosphorylase activity and cAMP generation was found to decrease with increasing age. The age-related changes in the pattern of stimulation of glycogen phosphorylase by catecholamines in isolated hepatocytes were accompanied by parallels changes in the numbers of alpha 1- and beta-adrenoceptors in membranes prepared from the isolated hepatocytes. A progressive decrease in the total number of alpha 1-receptors measured with [3H]-prazosin and beta-receptors measured with [125I]cyanopindolol (CYP) was found with increasing age. The loss of beta-adrenergic receptors was much more dramatic. Our results suggest that age-related alterations of hepatic glycogen phosphorylase activation by catecholamines may in part be explained by the changes in the expression adrenergic receptors.     

Acute effects of C-peptide on the microvasculature of isolated perfused skeletal muscles and kidneys in rat

The C-peptide has recently been suggested to have beneficial effects in several organs and improve glycaemic control in human type I diabetes, while there were no such effects in healthy controls. The exact mechanisms behind these effects are, however, not clear. In an attempt to study the actions of C-peptide on the microvasculature in normal rats during more controlled conditions, isolated rat hindquarters and kidneys were perfused with albumin solutions in order to obtain low basal concentrations of C-peptide. In rat hindquarters, infusion of C-peptide significantly increased the capillary filtration coefficients (CFC) from 0.035±0.002 to 0.044±0.002 mL min^-1 100 g^-1 mmHg^-1 (P<0.001, n=9) and the permeability surface area product (PS) for vitamin B₁₂ from 3.48±0.29 to 4.02±0.37 mL min^-1 100 g^-1 (P<0.01, n=6). Addition of C-peptide to the perfusate during infusion of sodium nitroprusside did not induce any additional alteration of CFC or PS. The vascular resistance was slightly decreased from 2.74±0.17 to 2.64±0.17 mmHg min 100 g mL^-1 (P<0.01, n=9). These effects of C-peptide are compatible with increases in capillary surface area without alteration of the permeability per se. In isolated rat kidneys perfused at low temperature (8 °C) prepared to inhibit all metabolic processes, C-peptide induced no changes in glomerular filtration rate, total vascular resistance or fractional albumin clearance. Therefore, C-peptide causes active vasodilation of the normothermic microvasculature and hence recruitment of capillaries. These findings support the previous observations in man that C-peptide indeed has biological effects.     

Glucose 6-phosphatase and glycogen phosphorylase activities in chondrocytes in epiphyseal cartilage of growing rats

Glycogen, glycogen phosphorylase, and glucose 6-phosphatase (G6Pase) activities were examined cytochemically in chondrocytes of femoral epiphyseal cartilages and cartilaginous ribs of 3- and 7-day-old rats. G6Pase activity was also examined biochemically. Glycogen was abundant in chondrocytes of the reserve zone, while it became scarce in the cells of the proliferative zone. From the upper part (adjoining the proliferative zone) to the lower part of the hypertrophic zone, glycogen accumulated in chondrocytes and decreased in the cells of the degenerative zone. Inversely, glycogen phosphorylase a and G6Pase activities were relatively high in chondrocytes of the proliferative zone and upper hypertrophic zone and were low in the cells of the reserve zone, lower hypertrophic zone, and degenerative zone. The reaction product for G6Pase was present in the endoplasmic reticulum and nuclear envelope of all types of chondrocytes composing the cartilages, although the amounts of reaction product varied with the cell types in parallel with the histochemical results. Biochemical G6Pase activity was higher in epiphyseal cartilages than in cartilaginous ribs. The possible mechanism and significance of the accumulation and decrease of glycogen in chondrocytes of the epiphyseal cartilage were discussed.     

Improved insulin‐stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation

Aim: Effects of in vivo adrenaline infusion on subsequent insulin‐stimulated glucose uptake and glycogen synthase activation was investigated in slow‐twitch (soleus) and fast‐twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. Methods: Male Wistar rats received adrenaline from osmotic mini pumps (≈150 μg kg^?1 h^?1) for 1 or 12 days before muscles were removed for in vitro studies. Results: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin‐stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin‐stimulated PKB ser⁴⁷³ phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin‐stimulated PKB phosphorylation was normal in both muscles. Conclusion: Insulin‐stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin‐stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.