Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by31P-and13C-MRS

The main aim of this study was to examine the hypothesis that creatine (Cr) feeding enhances myocellular glycogen storage in humans undergoing carbohydrate loading. Twenty trained male subjects were randomly assigned to have their diets supplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CRGP, n=10) or placebo (PL-GP, n=10) for 5 days. Changes in resting myocellular glycogen and phosphocreatine (PCr) were determined with Magnetic Resonance Spectroscopy (¹³C- and³¹P-MRS, respectively). After CR-GP, the levels of intramyocellular glycogen increased from 147±13 (standard error) mmol·(kg wet weight)^?1) to 182±17 mmol·(kg wet weight)^?1, while it increased from 134±17 mmol·(kg wet weight)^? to 182±17 mmol·(kg wet weight)^?1 after PL-GP; the increments in intramyocellular glycogen concentrations were not statistically different. The increment in the PCr/ATP ratio after CR-GP (+0.20±0.12) was significantly different compared to PL-GP (?0.34±0.16) (p<0.05). The present results do not support the hypothesis that Cr loading increases muscle glycogen storage.     

Changes in lipid and protein during starvation and the moulting cycle in the tiger prawn, Penaeus esculentus Haswell

Tiger prawns, Penaeus esculentus Haswell (mean wt 20.8 ± 0.3 g, range 13.9–27.7 g) contained 1–2% extractable lipid, 13% protein (biuret method) and 71–74% water (wet wt). In 21 days, the weight of fed prawns increased by 3% and that of starved prawns decreased by 4.4%. Protein was the major energy source during 14 days of starvation, with a loss of 550 mg of total protein compared with a loss of 84 mg of total lipid. The absolute amount of water present remained constant. Of three different tissue compartments, abdomen, cephalothorax, and digestive gland, the abdomen contributed the most protein (330 mg) and lipid (35 mg) during 14 days of starvation. Digestive gland, although containing the largest percentage wet wt of lipid, accounted for only 8.3% of the total lipid in the prawn, and contributed only 18 mg of lipid in 14 days of starvation. Lipid concentration in the digestive gland increased during early premoult (stage D₄) and dropped in late premoult (stage D₄). Resting oxygen consumption rate remained constant at ≈0.1 ml · g^?1 · h^?1 at 25°C during 21 days of starvation.     

Age-related augmentation of phosphorylase b kinase in hepatic tissue from the glycogen-storage-disease (gsd/gsd) rat.

The effects of food deprivation on body weight, liver weight, hepatic glycogen content, glycogenolytic enzymes and blood metabolites were compared in young and old phosphorylase b kinase-deficient (gsd/gsd) rats. Although the concentration of glycogen in liver from 9-week-old female gsd/gsd rats (730 mumol of glucose equivalents/g wet wt.) was increased by 7-8% during starvation, total hepatic glycogen was decreased by 12% after 24 h without food. In 12-month-old male gsd/gsd rats the concentration of liver glycogen (585 mumol of glucose equiv./g wet wt.) was decreased by 16% and total hepatic glycogen by nearly 40% after food deprivation for 24 h. Phosphorylase b kinase and phosphorylase a were present at approx. 10% of the control activities in 9-week-old gsd/gsd rats, but both enzyme activities were increased more than 3-fold in 12-month-old affected rodents. It is concluded that the age-related ability to mobilize hepatic glycogen appears to result from the augmentation of phosphorylase b kinase during maturation of the gsd/gsd rat.     

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy reserves during food deprivation and compensatory growth in juvenile roach: the importance of season and temperature

The effect of 21 days of starvation, followed by a period of compensatory growth during refeeding, was studied in juvenile roach Rutilus rutilus during winter and summer, at 4, 20 and 27° C acclimation temperature and at a constant photoperiod (12L : 12D). Although light conditions were the same during summer and winter experiments and fish were acclimated to the same temperatures, there were significant differences in a range of variables between summer and winter. Generally winter fish were better prepared to face starvation than summer fish, especially when acclimated at a realistic cold season water temperature of 4° C. In winter, the cold acclimated fish had a two to three‐fold larger relative liver size with an approximately double fractional lipid content, in comparison to summer animals at the same temperature. Their white muscle protein and glycogen concentration, but not their lipid content, were significantly higher. Season, independent of photoperiod or reproductive cycle, was therefore an important factor that determined the physiological status of the animal, and should generally be taken into account when fish are acclimated to different temperature regimes. There were no significant differences between seasons with respect to growth. Juvenile roach showed compensatory growth at all three acclimation temperatures with maximal rates of compensatory growth at 27° C. The replenishment of body energy stores, which were utilized during the starvation period, was responsible for the observed mass gain at 4° C. The contribution of the different energy resources (protein, glycogen and lipid) was dependent on acclimation temperature. In 20 and 27° C acclimated roach, the energetic needs during food deprivation were met by metabolizing white muscle energy stores. While the concentration of white muscle glycogen had decreased after the fasting period, the concentrations of white muscle lipid and protein remained more or less constant. The mobilization of protein and fat was revealed by the reduced size of the muscle after fasting, which was reflected in a decrease in condition factor. At 20° C, liver lipids and glycogen were mobilized, which caused a decrease both in the relative liver size and in the concentration of these substrates. Liver size was also decreased after fasting in the 4° C acclimated fish, but the substrate concentrations remained stable. This experimental group additionally utilized white muscle glycogen during food deprivation. Almost all measured variables were back at the control level within 7 days of refeeding.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

FACTORS AFFECTING THE TURNOVER OF CEREBRAL GLYCOGEN AND LIMIT DEXTRIN IN VIVO

The turnover of cerebral glycogen in mice has been investigated by using [U-¹⁴C]glucose as a precursor. The time required for turnover of total glycogen and limit dextrin has been determined in normal animals and animals given phenobarbital or hydrocortisone. In all 3 groups, the turnover time for limit dextrin was twice that of total glycogen. Phenobarbital increased the time for turnover of total glycogen and limit dextrin approximately 2-fold, whereas hydrocortisone diminished the turnover time of both fractions to one-half. The accumulation of glycogen during phenobarbital anesthesia (2·5-fold) is attributed to the decrease in rate of phosphorolysis rather than elevated glycogenesis. The ratio of phosphorylase a to total phosphorylase was significantly decreased in the brains of phenobarbital-treated mice, while the ratio of glycogen synthetase I to total synthetase activity was not affected. The administration of hydrocortisone had no effect on either the phosphorylase or synthetase of mouse brain. A mathematical model was devised to determine the rate constants for incorporation of labelled glucose into brain glycogen and the subsequent loss of radioactivity. Metabolite levels and enzyme activities have been correlated with the observed changes in glycogen turnover.     

Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

1. Hydrocortisone increases in vivo incorporation of [14C] glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a but was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19-21, and was without effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism 1 h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found without any change in synthetase a activity.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

 Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28–30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76±0.78 mg·g⁻¹ wet tissue in normal unexposed rats; 15.82±2.30 mg·g⁻¹ wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹) in comparison to normal (1.53 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure. Received: 30 March 1998 / Revised: 18 November 1998 / Accepted: 25 November 1998     

Inability of glucagon to regulate glycogen metabolism in rat hepatocytes isolated after fasting and refeeding high-carbohydrate diets

Studies are described which demonstrate that the ability of glucagon, epinephrine, and dibutyryl-cAMP to stimulate glycogenolysis is impaired in rat hepatocytes isolated from animals starved for 24 h and then refed a sucrose-rich diet or refed standard rat chow. The impaired regulation of glycogenolysis by glucagon was observed within 24 h after refeeding and persisted for at least 3 days. The inability of glucagon to stimulate glycogen breakdown in the refed condition appeared to be due to a suppressed activation of glycogen phosphorylase and phosphorylase b kinase by the hormone. The capacity of glucagon to regulate pyruvate kinase and glycolysis was not altered by refeeding, suggesting that the defect lies beyond interaction of the hormone at its receptor. Prolonged incubation of hepatocytes from refed rats was accompanied by depletion of glycogen reserves and was accompanied by restoration of hormonal stimulation of glycogenolysis. Addition of glycogen to cell-free extracts was found to inhibit phosphorylase b kinase but not phosphorylase. The findings of this investigation are consistent with the interpretation that high levels of glycogen present of liver after refeeding may lead to a diminished activity of phosphorylase b kinase and its hormonal regulation.     

Glycogen metabolizing enzymes during starvation and feeding of Ascaris suum maintained in a perfusion chamber

The glycogen content of muscle was correlated with the activity of glycogen synthase and glycogen phosphorylase from the parasitic roundworm Ascaris suum maintained in vitro. Adult female worms were maintained in the laboratory in a perfusion system during periods of starvation and feeding. During starvation, the levels of glucogen decreased at a rate of 0.1 to 0.2 mumoles/min/g wet weight of muscle-cuticle. During this time, 95% of the glycogen synthase (E.C. 2.4.1.11) was in the active D-form, and 48% of the phosphorylase (E.C. 2.4.1.1) was in the active a-form. Upon feeding, the rate of incorporation of glycosyl residues into glycogen proceeded at a rate of 0.75 to 1.0 mumoles/min/g muscle-cuticle. Glycogen synthase was 22% in the active I-form and phosphorylase a-levels remained virtually unchanged at 41% as compared with the starved worm. Total levels of both enzymes remained constant over the starvation-feeding period with 3.9 units/g phosphorylase and 0.4 units/g glycogen synthase. The apparent Km value for the substrate UDPG for glycogen synthase was 0.22 +/- 0.02 mM. For glycogen phosphorylase the Km value for G-1-P was 1.76 +/- 0.38 mM.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Daily rhythms of glycogen synthetase and phosphorylase activities in rat liver: Influences of food and light

In rats fed during the night time (from 8 p.m. to 8 a.m.) the activities of liver glycogen synthetase $\text{I}$ and phosphorylase $\text{a}$ varied rhythmically during a 24 hour period. There was an inverse relationship between their levels; the level of synthetase $\text{I}$ rose to a maximum at around 6 a.m. and that of phosphorylase $\text{a}$ attained the peak value at around 6 p.m. Eye enucleation of rats did not affect significantly the daily rhythms of the enzymes. However, when food was offered only during the day time, the phases of both enzyme rhythms were shifted by about 12 hours. On starvation for 24 hours, the glycogen level was reduced almost to nil, but the daily rhythms of the enzymes were retained. It is thus very likely that the daily variations of the enzyme activities are not merely a passive effect of food intake, and that food can be a synchronizer or zeitgeber which sets up the characteristic rhythms of glycogen metabolism in the liver.     

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.