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PubMed1990

Glycogen metabolism in cultured chick hepatocytes: a morphological study.

Ultrastructural and autoradiographic observations of cultured chick hepatocytes under the following conditions are described: Induction of glycogen synthesis with glucose alone and glucose plus insulin, and glucagon-induced glycogen breakdown. Profiles of hepatocytes cultured in medium containing 10 mM glucose showed typical cellular organelles and occasionally a few glycogen granules. After incubation of hepatocytes with 3H-glucose, silver grains were found over these sparse glycogen granules, indicating a low level of glycogen synthesis by a few cells. After addition of 75 mM glucose for 1 hr about 3% of the profiles of cells showed glycogen, and by 24 hr half of the hepatocytes had glycogen. Addition of insulin plus glucose induced glycogen synthesis in 82% of the cells after 6 hr, and by 24 hr almost every cellular profile showed glycogen particles. Morphologically, glycogen accumulation was similar whether the cells were stimulated by high glucose or by glucose plus insulin: glycogen granules appeared in restricted regions of the cytoplasm, which were rich in smooth endoplasmic reticulum (SER), and peroxisomes were found close to the newly deposited glycogen particles. At maximum glycogen accumulation the association of SER and peroxisomes with glycogen was less obvious. Glycogenolysis induced by incubation of glycogen-rich hepatocytes with glucagon resulted in proliferation of SER in the glycogen regions of the cells. These observations are compatible with the concept of regions in the hepatocyte cytoplasm specialized for glycogen metabolism. Possible roles for SER and peroxisomes found near glycogen particles and other organelles in hepatic glycogen metabolism are discussed.

Animals↗
PubMed1991

Muscle glycogen synthesis before and after exercise.

The importance of carbohydrates as a fuel source during endurance exercise has been known for 60 years. With the advent of the muscle biopsy needle in the 1960s, it was determined that the major source of carbohydrate during exercise was the muscle glycogen stores. It was demonstrated that the capacity to exercise at intensities between 65 to 75% VO2max was related to the pre-exercise level of muscle glycogen, i.e. the greater the muscle glycogen stores, the longer the exercise time to exhaustion. Because of the paramount importance of muscle glycogen during prolonged, intense exercise, a considerable amount of research has been conducted in an attempt to design the best regimen to elevate the muscle's glycogen stores prior to competition and to determine the most effective means of rapidly replenishing the muscle glycogen stores after exercise. The rate-limiting step in glycogen synthesis is the transfer of glucose from uridine diphosphate-glucose to an amylose chain. This reaction is catalysed by the enzyme glycogen synthase which can exist in a glucose-6-phosphate-dependent, inactive form (D-form) and a glucose-6-phosphate-independent, active form (I-form). The conversion of glycogen synthase from one form to the other is controlled by phosphorylation-dephosphorylation reactions. The muscle glycogen concentration can vary greatly depending on training status, exercise routines and diet. The pattern of muscle glycogen resynthesis following exercise-induced depletion is biphasic. Following the cessation of exercise and with adequate carbohydrate consumption, muscle glycogen is rapidly resynthesised to near pre-exercise levels within 24 hours. Muscle glycogen then increases very gradually to above-normal levels over the next few days. Contributing to the rapid phase of glycogen resynthesis is an increase in the percentage of glycogen synthase I, an increase in the muscle cell membrane permeability to glucose, and an increase in the muscle's sensitivity to insulin. The slow phase of glycogen synthesis appears to be under the control of an intermediate form of glycogen synthase that is highly sensitive to glucose-6-phosphate activation. Conversion of the enzyme to this intermediate form may be due to the muscle tissue being constantly exposed to an elevated plasma insulin concentration subsequent to several days of high carbohydrate consumption. For optimal training performance, muscle glycogen stores must be replenished on a daily basis. For the average endurance athlete, a daily carbohydrate consumption of 500 to 600g is required. This results in a maximum glycogen storage of 80 to 100 mumol/g wet weight.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary Carbohydrates↗
PubMedDate not supplied

Placental glycogen metabolism in diabetic pregnancy.

Glycogen content in the normal placenta decreases gradually towards term. However, in human diabetes and in rat streptozotocin diabetes two- to tenfold increases in placental glycogen level were found during the pregnancy. This elevation was evident in rats per tissue weight, protein or DNA content and was also seen in insulin-treated and gestational diabetics. Electron microscopic investigation of diabetic rat placenta revealed glycogen deposition in the typical glycogen cells, also in junctional zone cells and in all cells of the placental labyrinth. Placental glycogen accumulation in diabetes occurs in marked contrast to other tissues, such as maternal liver, from which glycogen disappears. Liver and muscle glycogenesis and glycogenolysis are under insulin control, by regulation of the activities of glycogen synthase and phosphorylase. However, in the placenta these enzymes are not meaningfully influenced by insulin in in vivo and in vitro studies. In our and other laboratories the activities of both enzymes somewhat increased or decreased, showing no trend conducive to glycogen accumulation. Placenta is glucose dependent, but the role of insulin in its carbohydrate metabolism is doubtful. Despite the high placental concentration of insulin receptors no metabolic outcome has yet been pointed out. Glycogen accumulation in the placenta of diabetic rats was found to be related to the extent of maternal hyperglycemia. The resultant markedly increased intracellular level of glucose-6-phosphate accelerates glycogen synthesis b. Glucose itself activates glycogen synthase and deactivates glycogen phosphorylase. Continuous glucose infusion to non-diabetic pregnant rats on gestation days 18-21 likewise also caused an increase in placental glycogen in correlation with hyperglycemia. The possibility that placental glycogen is under the control of fetal rather than maternal insulin was explored by producing insulin deficiency through intrafetal streptozotocin injection. There was no effect of fetal "diabetes" on placental glycogen synthesis or on the distribution of placental glycogen between the maternal and fetal segments of the placenta, while it caused a marked decrease in the fetal liver glycogen content and fetal body weight. To assess the availability of placental glycogen as an energy source the placental glycogenolysis was investigated after hormonal stimulation. Catecholamines were effective in inducing lactate formation both in vivo and in vitro in nondiabetic and diabetic rats. Protracted activation of the adenylate cyclase system by cholera toxin administration pronouncedly reduced placental glycogen in vivo.

Animals↗
PubMed1988

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes.

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.

Animals↗
PubMed1997

Exercise in transgenic mice overexpressing GLUT4 glucose transporters: effects on substrate metabolism and glycogen regulation.

We assessed the effects of GLUT4 glucose transporter expression on substrate metabolism and glycogen regulation during exercise. Transgenic mice overexpressing human (h)GLUT4 in muscle and fat (TG) and their wild-type littermates (WT) were studied by indirect calorimetry at rest and during acute treadmill exercise (30 minutes) and recovery (30 minutes). The rate of carbon dioxide production (VCO2) increased to a greater degree in TG during exercise, whereas resting VCO2, resting oxygen production (VO2), and exercise-induced increments in VO2 were similar in TG and WT. As a result, the respiratory quotient (RQ) was increased by .03 to .05 in TG during exercise, due to greater consumption of carbohydrate (up to approximately 64% more) and less consumption of lipid (up to approximately 40% less) compared with WT, without differences in overall energy expenditure. These differences in substrate metabolism were observed despite relative hypoglycemia and elevated free fatty acids (FFAs) in TG that persisted throughout resting, exercise, and recovery periods. To further assess substrate availability, glycogen content and glycogen synthase activity were measured in skeletal muscle and liver. At rest, muscle glycogen content was 50% higher and glycogen synthase I was 40% lower in TG compared with WT. During exercise and recovery, muscle glycogen was more profoundly depleted in TG than in WT, and glycogen synthase I increased to levels observed in WT, with no change in total glycogen synthase. In the liver, glycogen content and total glycogen synthase were similar in TG and WT under resting conditions, while glycogen synthase I was reduced by 48%. Exercise and recovery induced a more profound depletion of liver glycogen (76% v 30%) and greater increments in both I-form and total glycogen synthase in TG. In conclusion, (1) TG overexpressing GLUT4 exhibit greater muscle glycogen content at rest than WT; (2) during exercise, TG metabolize more carbohydrate, made possible by increased glycogenolysis in muscle and liver, and this predominates as a fuel source despite hypoglycemia and increased availability of FFA; (3) increased carbohydrate metabolism is linked to a decrease in lipid metabolism such that there is no change in overall energy expenditure; and (4) glycogen synthase I activity is inversely proportional to tissue glycogen content despite differences in circulating glucose, insulin, and FFA concentrations, indicating that glycogen content has an overriding regulatory influence on glycogen synthase.

Animals↗
PubMed1976

Quantitative cytochemistry of glycogen in blood cells. Methods and clinical application.

Quantitative glycogen determinations can be made in single blood and bone marrow cells, using microspectrophotometry or microfluorometry after staining with variants of the periodic acid--Schiff (PAS) reaction. These PAS variant reactions generally do not indicate the presence of non-glycogen PAS-positive substances, known to be prevalent in various hematopoietic cells, possibly due to masking of reactive groups. The specificity of the reaction in blood cells was ascertained by alpha-amylase digestion, which removed more than 95% of the PAS-positive material. Calibration of the PAS reaction was undertaken with a microdroplet model of pure leukocyte glycogen. The glycogen amounts in the droplets were determined by microinterferometry, the droplets were stained with a variant PAS reaction, and the total extinction of the reaction product in the stained droplets was determined by microspectrophotometry. The extinction coefficient (k) was obtained from the equation k equals Etot divided by M where (Etot) is the total extinction as determined by microspectrophotometry and (M) the dry glycogen amount as determined by microinterferometry. The microinterferometric dry mass determinations were calibrated by X-ray absorption in order to obtain the absolute amounts of glycogen. For practical purposes a reference system was made of normal neutrophil leukocytes. The glycogen content in the reference neutrophils was first determined with the micromodel. These neutrophils, now with a known glycogen amount, were stained with the PAS reagents and measured microspectrophotometrically in parallel with cells containing an unknown glycogen amount. Alternatively, the staining was made with a fluorescent PAS reaction, and the glycogen content determined by microfluorometry. Both methods appeared suitable for determining the glycogen content of blood cells from patients with various diseases, though the microfluorometric method was preferable for measurements of small amounts of inhomogeneously distributed glycogen. The mean glycogen content of normal neutrophil leukocytes was found to be 13.6 times 10(-12) g. The content was increased in infectious diseases such as pneumonia and tonisillitis, as well as in polycythemia vera and myelofibrosis, while low amounts were found in untreated chronic myelocytic leukemia. In chronic myelocytic leukemia in remission, the glycogen content of mature neutrophils had completely normalized. Erythroblasts normally do not contain detectable amounts of glycogen. However, in certain diseases such as beta-thalassemia and Di Guglielomo's syndrome, appreciable amounts of glycogen accumulate in the erythropoietic precursor cells. In beta-thalassemia this was associated with an arrest in the proliferation of early polychromatic erythroblasts, which accumulate glycogen in the G1 phase of the cell cycle. In all these diseases quantitative glycogen determinations in the blood cells have diagnostic importance.

Autoradiography↗
PubMed1985

Mechanism of liver glycogen repletion in vivo by nuclear magnetic resonance spectroscopy.

In order to quantitate the pathways by which liver glycogen is repleted, we administered [1-13C]glucose by gavage into awake 24-h fasted rats and examined the labeling pattern of 13C in hepatic glycogen. Two doses of [1-13C]glucose, 1 and 6 mg/g body wt, were given to examine whether differences in the plasma glucose concentration altered the metabolic pathways via which liver glycogen was replenished. After 1 and 3 h (high-dose group) and after 1 and 2 h (low-dose group), the animals were anesthetized and the liver was quickly freeze-clamped. Liver glycogen was extracted and the purified glycogen hydrolyzed to glucose with amyloglucosidase. The distribution of the 13C-label was subsequently determined by 13C-nuclear magnetic resonance spectroscopy. The percent 13C enrichment of the glucosyl units in glycogen was: 15.1 +/- 0.8%(C-1), 1.5 +/- 0.1%(C-2), 1.2 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 1.6 +/- 0.1%(C-5), and 2.2 +/- 0.1%(C-6) for the high-dose study (n = 4, at 3 h); 16.5 +/- 0.5%(C-1), 2.0 +/- 0.1%(C-2), 1.3 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 2.2 +/- 0.1%(C-5), and 2.4 +/- 0.1%(C-6) in the low-dose study (n = 4, at 2 h). The average 13C-enrichment of C-1 glucose in the portal vein was found to be 43 +/- 1 and 40 +/- 2% in the high- and low-dose groups, respectively. Therefore, the amount of glycogen that was synthesized from the direct pathway (i.e., glucose----glucose-6-phosphate----glucose-1-phosphate----UDP-glucose---- glycogen) was calculated to be 31 and 36% in the high- and low-dose groups, respectively. The 13C-enrichments of portal vein lactate and alanine were 14 and 14%, respectively, in the high-dose group and 11 and 8%, respectively, in the low-dose group. From these enrichments, the minimum contribution of these gluconeogenic precursors to glycogen repletion can be calculated to be 7 and 20% in the high- and low-dose groups, respectively. The maximum contribution of glucose recycling at the triose isomerase step to glycogen synthesis (i.e., glucose----triose-phosphates----glycogen) was estimated to be 3 and 1% in the high- and low-dose groups, respectively. In conclusion, our results demonstrate that (a) only one-third of liver glycogen repletion occurs via the direct conversion of glucose to glycogen, and that (b) only a very small amount of glycogen synthesis can be accounted for by the conversion of glucose to triose phosphates and back to glycogen; this suggests that futile cycling between fructose-6-phosphate and fructose-1,6-diphosphate under these conditions is minimal. Our results also show that (c) alanine and lactate account for a minimum of between 7 and 20% of the glycogen synthesized, and that (d) the three pathways through which the labeled flux is measured account for a total of only 50% of the total glycogen synthesized. These results suggest that either there is a sizeable amount of glycogen synthesis via pathway(s) that were not examined in the present experiment or that there is a much greater dilution of labeled alanine/lactate in the oxaloacetate pool than previously appreciated, or some combination of these two explanations.

Alanine↗
PubMed1990

Substrate cycling between glucose 6-phosphate and glycogen occurs in Schistosoma mansoni.

The regulation of glycogen metabolism in Schistosoma mansoni was studied in vitro with special emphasis on the possible occurrence of substrate ('futile') cycling. The partition of label between carbon atoms 1 and 6 of the glucose units in glycogen was analysed after the incubation of intact worm pairs in the presence of [6-14C]glucose. Under all conditions tested, more than 99% of the label in glycogen was still in the 6 position, demonstrating that glycogen was synthesised not via an indirect pathway involving 3-carbon units, but directly, from glucose. Increasing the glucose concentration stimulated glycogen synthase and decreased the activity of glycogen phosphorylase. An inverse relationship was shown between the actual glycogen content and the rate of glycogenesis. Substrate cycling occurred between glucose 6-phosphate and glycogen. Glucose was incorporated into glycogen during periods of net glycogen breakdown, and vice versa: glycogen degradation occurred during periods of net glycogen synthesis. Under our experimental conditions of net glycogen degradation, the rate of glycogen synthesis as a percentage of that of glycogen breakdown was dependent on the external glucose concentration and ranged from 5 to 68% for 2 to 100 mM glucose, respectively. The synthesis of glycogen during periods of net glycogen breakdown was shown to occur in each individual worm pair.

Animals↗
PubMed1994

Inhibition of glycogen synthesis by epidermal growth factor in hepatocytes. The role of cell density and pertussis toxin-sensitive GTP-binding proteins.

Epidermal growth factor (EGF) counteracts the stimulation of glycogen synthesis by insulin in hepatocytes, but it is not known whether this is due to inhibition of glycogen synthesis or to inhibition of the insulin-signalling mechanism. This study investigates the mechanisms by which EGF affects the basal rate and the insulin stimulation of glycogen synthesis. The basal rate of glycogen synthesis is higher at low than at high cell density. EGF inhibits the basal rate of glycogen synthesis at low cell density but not in confluent cultures and abolishes the difference due to density. However, EGF inhibits the stimulation of glycogen synthesis by insulin irrespective of cell density. Increasing glycogen synthesis by increasing the [glucose] does not abolish the difference in rates of glycogen synthesis due to cell density, neither does it induce responsiveness to EGF at high cell density, establishing that responsiveness to EGF is a function of cell density and not of the basal rate and that inhibition of the insulin stimulation also cannot be accounted for by the higher rate of glycogen synthesis. Cytochalasin D and phalloidin, which alter cell morphology through interactions with the microfilament cytoskeleton, mimic the cell-density-dependent inhibition of glycogen synthesis by EGF. The inhibition of glycogen synthesis by EGF and cytochalasin D is additive and cytochalasin D potentiates the inhibition of glycogen synthesis by EGF, suggesting involvement of a cytoskeletal mechanism. Exogenous phospholipase C inhibits glycogen synthesis at both low and high cell density and the inhibition at low cell density is not additive with that caused by either EGF or cytochalasin D, suggesting that these agonists inhibit glycogen synthesis through changes in Ca2+ and/or diacylglycerol. The inhibition of glycogen synthesis by EGF in the absence of insulin stimulation is blocked by neomycin, which inhibits Ca2+ release from intracellular stores but not by antagonists of protein kinase C. It was also inhibited by pertussis toxin (50%), suggesting that it may involve GTP-binding-protein-mediated release of Ca2+ from intracellular stores. The inhibition of the stimulation of glycogen synthesis by insulin was not affected by neomycin and was only marginally inhibited by pertussis toxin or guanosine 5'-O-[3-thio]triphosphate (GTP[S]). We infer from these findings that the inhibition by EGF of the basal rate of glycogen synthesis and of the insulin stimulation are mediated by different mechanisms. The latter is pertussis toxin insensitive and independent of cell density, whereas the former is expressed only at low cell density, it is potentiated by cytochalasin D and inhibited by pertussis toxin.

Animals↗
PubMed1986

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers.

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.

Animals↗
PubMed1995

Glycogen concentration and regulation of synthase activity in rat liver in vivo.

The proportion of liver glycogen synthase in the active (R+I) forms is lower in the fed than in the fasted state. This has been attributed to inhibition of synthase phosphatase by the accumulated hepatic glycogen. We observed that after oral administration of glucose or galactose to fasted rats, hepatic synthase R+I activity first increased, as expected, but then decreased to a nadir significantly below the control level regardless of the maximal glycogen concentration reached. Therefore, we investigated further the relationship of hepatic synthase R+I activity and glycogen concentration in vivo in fasted rats given increasing oral glucose loads. Male rats fasted 24 h were given glucose doses of 0.1-4.0 g/kg by oral gavage (n > or = 8). Liver synthase R+I, total synthase, phosphorylase a, glucose, glycogen, glucose-6-P, and UDP glucose were measured at intervals over 20-240 min after gavage. Even the smallest glucose load elicited rapid glycogen synthesis. The maximum glycogen concentration increased linearly with the size of the glucose load, although the proportion of administered glucose accounted for by liver glycogen decreased as the glucose load increased. The proportion of synthase in the active (R+I) forms peaked at 20 min after all doses and then declined. By the time the glycogen concentration was maximal, synthase R+I activity had decreased to the control value, regardless of the glucose dose administered or the maximum glycogen concentration reached. Although the decrease in synthase R+I at the time of the glycogen peak was correlated with the increase in glycogen concentration, synthase R+I continued to decrease for another 1-2 h even though liver glycogen was stable or decreasing. The nadir reached was independent of the maximal glycogen concentration. The synthase R+I nadir also did not correlate with hepatic glucose or glucose-6-P concentrations or phosphorylase a activity. Overall, there was not a straightforward temporal or quantitative relationship between the glycogen concentration and synthase R+I activity. These data suggest a more complex mechanism than simply direct inhibition of synthase phosphatase by glycogen.

Animals↗
PubMed1980

Kinetic mechanism of rabbit muscle glycogen synthase I.

The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

Animals↗
PubMed1992

Regulation of glycogen synthesis and glycolysis by insulin, pH and cell volume. Interactions between swelling and alkalinization in mediating the effects of insulin.

The effects of changes in cell volume and pH on glycogen synthesis and glycolysis and their control by insulin were investigated in hepatocyte cultures. 1. Cell acidification, by increasing [CO2] from 2.5% to 5%, inhibited glycolysis and stimulated glycogen synthesis. The inhibition of glycolysis was also observed in Na(+)-free media and when K+ uptake was inhibited, but the stimulation of glycogen synthesis was abolished under these conditions, suggesting that it is secondary to ionic or volume changes. Alkalinization had converse effects on glycolysis and glycogen synthesis. 2. In HCO3(-)-containing media, replacement of NaCl with sodium acetate or potassium acetate, like acidification with CO2, inhibited glycolysis and stimulated glycogen synthesis. The latter correlated with an increase in cation content. Amiloride, an inhibitor of Na+/H+ exchange, inhibited both the increase in cation content and the stimulation of glycogen synthesis, suggesting that the latter is secondary to cell swelling. 3. Hypo-osmotic swelling increased glycogen synthesis in HCO3(-)-containing media, in both the absence and the presence of Na+ and at both 2.5% and 5% CO2, but it increased glycolysis in the presence of Na+ and at 2.5%, but not at 5%, CO2. In HCO3(-)-free media, during acidification and swelling, glycogen synthesis correlated with pH and not with cell volume, indicating that inhibition by acidification over-rides stimulation by swelling. 4. Stimulation of glycolysis by insulin was not additive with stimulation by alkalinization. The stimulation of glycogen synthesis by insulin was partially suppressed under alkaline conditions; it was markedly suppressed in isosmolar Na(+)-free media and restored by hypo-osmotic swelling. In hypo-osmolar Na(+)-free media insulin prevented the decrease in glycogen synthesis with decreasing [HCO3-], suggesting that it counteracts inhibition by acidification. 5. It is concluded that glycogen synthesis and glycolysis are both stimulated by cell swelling and inhibited by acidification, under certain conditions, but glycolysis is more sensitive to inhibition by acidification and glycogen synthesis to stimulation by swelling. Consequently, simultaneous swelling and acidification is associated with inhibition of glycolysis and stimulation of glycogen synthesis. Stimuli that cause swelling and alkalinization activate both glycogen synthesis and glycolysis, alkalinization being more important in control of glycolysis and swelling in control of glycogen synthesis. Both cell swelling and alkalinization are components of the mechanism by which insulin controls glycogen synthesis and glycolysis.

Acetates↗
PubMed2001

Stimulating effects of low-dose fructose on insulin-stimulated hepatic glycogen synthesis in humans.

Fructose has been shown to have a catalytic effect on glucokinase activity in vitro; however, its effects on hepatic glycogen metabolism in humans is unknown. To address this question, we used (13)C nuclear magnetic resonance (NMR) spectroscopy to noninvasively assess rates of hepatic glycogen synthesis and glycogenolysis under euglycemic (approximately 5 mmol/l) hyperinsulinemic conditions (approximately 400 pmol/l) with and without a low-dose infusion of fructose (approximately 3.5 micromol. kg(-1). min(-1)). Six healthy overnight-fasted subjects were infused for 4 h with somatostatin (0.1 micromol. kg(-1). min(-1)) and insulin (240 pmol. m(-2). min(-1)). During the initial 120 min, [1-(13)C]glucose was infused to assess glycogen synthase flux followed by an approximately 120-min infusion of unlabeled glucose to assess rates of glycogen phosphorylase flux. Acetaminophen was given to assess the percent contribution of the direct and indirect (gluconeogenic) pathways of glycogen synthesis by the (13)C enrichment of plasma UDP-glucuronide and C-1 of glucose. In the control studies, the flux through glycogen synthase and glycogen phosphorylase was 0.31 +/- 0.06 and 0.17 +/- 0.04 mmol/l per min, respectively, and the rate of net hepatic glycogen synthesis was 0.14 +/- 0.05 mmol/l per min. In the fructose studies, the glycogen synthase flux increased 2.5-fold to 0.79 +/- 0.16 mmol/l per min (P = 0.018 vs. control), whereas glycogen phosphorylase flux remained unchanged (0.24 +/- 0.06; P = 0.16 vs. control). The infusion of fructose resulted in a threefold increase in rates of net hepatic glycogen synthesis (0.54 +/- 0.12 mmol/l per min; P = 0.008 vs. control) without affecting the pathways of hepatic glycogen synthesis (direct pathway approximately 60% in both groups). We conclude that during euglycemic hyperinsulinemia, a low-dose fructose infusion causes a threefold increase in net hepatic glycogen synthesis exclusively through stimulation of glycogen synthase flux. Because net hepatic glycogen synthesis has been shown to be diminished in patients with poorly controlled type 1 and type 2 diabetes, stimulation of hepatic glycogen synthesis by this mechanism may be of potential therapeutic value.

Adult↗
PubMed2004

Free fatty acids increase hepatic glycogen content in obese males.

Obesity is associated with increased hepatic glycogen content. In vivo and in vitro data suggest that plasma free fatty acids (FFA) may cause this increase. In this study we investigated the effect of physiological plasma FFA levels on hepatic glycogen metabolism by studying intrahepatic glucose pathways in lean and obese subjects. Six lean and 6 obese males were studied twice during a 16- to 22-hour fast, once with and once without acipimox, an inhibitor of lipolysis. Intrahepatic glucose fluxes were measured by infusion of [2-(13C1)]glycerol, [1-(2H1)]galactose, and [U-(13C6)]glucose. Acetaminophen was administered as a glucuronate probe. In both lean and obese control studies, plasma FFA levels increased progressively, whereas acipimox completely suppressed plasma FFA levels for the whole study period. In lean males glycogenolysis did not change in the acipimox study, but decreased in the control study (P < .01). In lean males, neither glycogen synthesis, glycogen synthesis retained as glycogen, nor glycogen balance differed between control and acipimox studies. In obese males glycogenolysis did not change in the acipimox study, but decreased in the control study (P < .01). Glycogen synthesis did not change in either study. Glycogen synthesis retained as glycogen did not change in acipimox study, but increased in the control study (P = .03). Glycogen balance did not change in the acipimox study, but increased in the control study (P < .01). This study demonstrates that in obese males physiological levels of FFA contribute to the retention of hepatic glycogen during short-term fasting by inhibiting breakdown of glycogen and increasing glycogen synthesis retained as glycogen, whereas in lean males this effect was absent due to unaltered glycogen synthesis retained as glycogen.

Adult↗
PubMed1982

Changes in liver lobule glycogen zonation during prolonged fasting of rats previously fed a 30% casein diet and adapted to a controlled feeding schedule.

Changes in the lobular distribution of liver glycogen were studied during the prolonged fasting of young adult male Sprague-Dawley rats that were previously adapted to a 30% casein diet and to the 2 + 22 controlled feeding and lighting schedule. The optical density of glycogen, revealed in fixed liver cryostat sections by the periodic acid-Schiff procedure, was determined in the periportal (P), midlobular (M), and centrilobular (C) regions of the liver lobule at various times during a 196-hour fasting period. The prolonged fast could be divided into 3 phases with respect to liver glycogen variation: initial glycogen depletion, glycogen resurgence, and final glycogen depletion. Glycogen was lost from all regions of the liver lobule during the initial glycogen depletion phase. During the glycogen resurgence phase, a lobular glycogen concentration gradient formed (P greater than M greater than C). The simultaneous occurrence of increasing liver glycogen, increasing liver tyrosine aminotransferase activity, and decreasing body fat during glycogen resurgence suggests that the young adult rat does not spare body protein during starvation. During the final glycogen depletion phase, liver glycogen was again present in a lobular gradient (P greater than M greater than C) but the absolute amount of glycogen in each region of the lobule was markedly reduced in comparison with rats killed during glycogen resurgence.

Adaptation, Physiological↗
PubMed1983

Ontogeny of some enzymes of glycogen metabolism in rabbit fetal heart, lungs, and liver.

Optimum conditions were established for the assay of glycogen, glycogen synthase, glycogen phosphorylase, phosphoglucomutase, and glucose-6-phosphatase in rabbit fetal heart, lung, and liver. Using these methods, the pattern of appearance of glycogen and the above four enzymes was established from day 18 of gestation to day 8 after birth. The results indicate that total tissue glycogen reaches maximum levels between days 22 and 24 in the heart, days 24 and 26 in the lung, and days 30 and 31 in the liver. In all three tissues, the rapid rise or depletion of glycogen is coincident with a corresponding increase in glycogen synthase and glycogen phosphorylase activities. However, substantial amounts of glycogen synthase are present both prior to and after the accumulation of glycogen. Similarly, considerable amounts of glycogen phosphorylase are present early in gestation, yet deposition of glycogen occurs. Both the I and D forms of glycogen synthase are present in the three tissues, the major being the physiologically inactive D form. Similarly both the a and b forms of glycogen phosphorylase are present, with the a form (active form) making up about 30-60% of the total phosphorylase activity. Glucose-6-phosphatase was absent in fetal heart and lung throughout the period of gestation investigated. Low levels of this enzyme were detectable in fetal liver near term. The phosphoglucomutase activity increased progressively from day 22 of gestation in all three tissues and continues to increase after birth. The disappearance of fetal lung glycogen occurs between days 27 and 28 at a time when surfactant phospholipids first appear. These findings indicate that the breakdown of glycogen is providing the fetal lung cells with energy necessary for surfactant phospholipid biosynthesis.

Animals↗
PubMed1996

Contribution of glycogen to aerobic myocardial glucose utilization.

BACKGROUND: We determined glycogen turnover and the contribution of glycogen as a source of glucose for aerobic myocardial glycolysis and glucose oxidation in parallel series of isolated, working rat hearts subjected to pulse-chase perfusions. METHODS AND RESULTS: Myocardial glycogen of isolated, working rat hearts was radiolabeled, after 30 minutes of substrate-free glycogen depletion, by perfusion for 60 minutes with buffer designed to stimulate resynthesis of glycogen (1.2 mmol/L palmitate, 11 mmol/L [U-14C]- or [5-3H]glucose, 0.5 mmol/L lactate, and 100 microU/mL insulin). Rates of glucose oxidation (14 CO2 production) and glycolysis (3H20 production) were then measured by perfusing the hearts for 40 minutes with buffer designed to simulate physiological conditions (0.4 mmol/L palmitate, 0.5 mmol/L lactate, 11 mmol/L [5-3H]- or [U-14C]- glucose, 100 microU/mL insulin) containing radiolabeled glucose different from that used during resynthesis. During the chase perfusion, rates of glycolysis and glucose oxidation from exogenous glucose were significantly greater than those from endogenous glycogen. Nevertheless, glycogen contributed significantly to myocardial energy production (41% of the overall ATP produced from glucose), and a significantly greater fraction of the glucose from glycogen that passed through glycolysis was oxidized (>50%) compared with the fraction oxidized from exogenous glucose (<20%, P<.05). Myocardial glycogen was simultaneously synthesized and degraded (ie, glycogen turnover) during the chase perfusion, despite net glycogenolysis. Furthermore, enrichment of labeled glucose in glycogen at the end of the chase perfusion, when corrected for newly synthesized glycogen, did not differ from that at the end of the labeling period. CONCLUSIONS: Thus, glycogen contributes significantly to aerobic myocardial glucose use under these experimental conditions, and the glucose derived from glycogen is oxidized preferentially compared with exogenous glucose. Additionally, substantial myocardial glycogen turnover occurs, and the manner in which glycogen is degraded does not fit the ordered hypothesis of "last glucose on, first glucose off."

Adenosine Triphosphate↗