scholarly journals Glycogen synthesis by rat hepatocytes

1979 ◽  
Vol 180 (2) ◽  
pp. 389-402 ◽  
Author(s):  
J Katz ◽  
S Golden ◽  
P A Wals
Keyword(s):  
Glycogen Phosphorylase ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Phosphorylated Form ◽  
Cell Pellet ◽  
Glycogen Deposition ◽  
Gluconeogenic Substrates ◽  
Total Enzyme

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12–15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50–60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.

Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes

2001 ◽  
Vol 358 (3) ◽  
pp. 665-671 ◽  
Author(s):  
Lori A. GUSTAFSON ◽  
Mies NEEFT ◽  
Dirk-Jan REIJNGOUD ◽  
Folkert KUIPERS ◽  
Hans P. SAUERWEIN ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Lactate Production ◽  
Rat Hepatocytes ◽  
Glycolytic Flux ◽  
Isolated Rat Hepatocytes ◽  
Glycogen Accumulation ◽  
Glycogen Deposition ◽  
Intracellular Glucose

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted → fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.

scholarly journals Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism

1997 ◽  
Vol 326 (3) ◽  
pp. 823-827 ◽  
Author(s):  
Vinciane GAUSSIN ◽  
Philippe GAILLY ◽  
Jean-Marie GILLIS ◽  
Louis HUE
Keyword(s):  
Glycogen Phosphorylase ◽  
Time Course ◽  
Buffer Capacity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Glycogen Metabolism ◽  
Dose Dependence ◽  
Ion Concentration ◽  
Extracellular Medium

In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 μmol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration, as measured with mag-fura 2, increased from 0.25 to 0.43 μmol/g of cells and 0.35 μmol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration.

scholarly journals Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes

1987 ◽  
Vol 248 (2) ◽  
pp. 429-437 ◽  
Author(s):  
A Lavoinne ◽  
A Baquet ◽  
L Hue
Keyword(s):  
Glycogen Phosphorylase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Rat Hepatocytes ◽  
Diabetic Rats ◽  
Purine Analogues ◽  
Isolated Rat Hepatocytes ◽  
Body Production ◽  
Stimulation Of

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.

scholarly journals Shared control of hepatic glycogen synthesis by glycogen synthase and glucokinase

2000 ◽  
Vol 351 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Roger R. GOMIS ◽  
Juan C. FERRER ◽  
Joan J. GUINOVART
Keyword(s):  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Shared Control ◽  
Hepatic Glycogen ◽  
Dependent Manner ◽  
Rate Limiting Step ◽  
Intermediate Metabolites ◽  
Cultured Rat Hepatocytes ◽  
Glycogen Deposition

We have used recombinant adenoviruses (AdCMV-RLGS and AdCMV-GK) to overexpress the liver isoforms of glycogen synthase (GS) and glucokinase (GK) in primary cultured rat hepatocytes. Glucose activated overexpressed GS in a dose-dependent manner and caused the accumulation of larger amounts of glycogen in the AdCMV-RLGS-treated hepatocytes. The concentration of intermediate metabolites of the glycogenic pathway, such as glucose 6-phosphate (Glc-6-P) and UDP-glucose, were not significantly altered. GK overexpression also conferred on the hepatocyte an enhanced capacity to synthesize glycogen in response to glucose, as described previously [Seoane, Gómez-Foix, O'Doherty, Gómez-Ara, Newgard and Guinovart (1996) J. Biol. Chem. 271, 23756–23760], although, in this case, they accumulated Glc-6-P. When GS and GK were simultaneously overexpressed, the accumulation of glycogen was enhanced in comparison with cells overexpressing either GS or GK. Our results are consistent with the hypothesis that liver GS catalyses the rate-limiting step of hepatic glycogen synthesis. However, hepatic glycogen deposition from glucose is submitted to a system of shared control in which the ‘controller’, GS, is, in turn, controlled by GK. This control is indirectly exerted through Glc-6-P, which ‘switches on’ GS dephosphorylation and activation.

Hepatic carbohydrate metabolism in the spontaneously diabetic Bio-Breeding Worcester rat

1981 ◽  
Vol 240 (2) ◽  
pp. E83-E87 ◽  
Author(s):  
M. C. Appel ◽  
A. A. Like ◽  
A. A. Rossini ◽  
D. B. Carp ◽  
T. B. Miller
Keyword(s):  
Carbohydrate Metabolism ◽  
Glycogen Phosphorylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glucose Utilization ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glucose Production ◽  
Juvenile Onset ◽  
Beta Cell Destruction

The effects of diabetes on hepatic carbohydrate metabolism were investigated in spontaneously diabetic Bio-Breeding Worcester (BB/W) rats. The juvenile-onset-type syndrome displayed by these animals is characterized by beta-cell destruction with subsequent ketosis-prone insulinopenia. Livers from diabetic animals demonstrated increased adenosine 3',5'-cyclic monophosphate levels but subnormal total protein and glycogen content. Isolated perfused livers of diabetic BB/W rats demonstrated an increased rate of glucose production from [14C]lactate and an impaired rate of glycogen synthesis. These data were consonant with hepatic enzyme studies demonstrating markedly increased activities of component gluconeogenic (glucose-6-phosphatase, fructose-1,6-diphosphatase, phosphoenolpyruvate carboxykinase) and glycogenolytic (glycogen phosphorylase) enzymes with decreased activities of glycolytic (hexokinase, pyruvate kinase) and glycogenic (glycogen synthase) enzymes. These findings agree with previous studies using alloxan- and streptozotocin-induced diabetic animals and suggest that accelerated hepatic gluconeogenesis and impaired glucose utilization are pathognomonic of all insulin-deficient diabetic syndromes.

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

2008 ◽  
Vol 294 (1) ◽  
pp. E28-E35 ◽  
Author(s):  
Michale Bouskila ◽  
Michael F. Hirshman ◽  
Jørgen Jensen ◽  
Laurie J. Goodyear ◽  
Kei Sakamoto
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Muscle Glycogen ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Wild Type ◽  
Muscle Glycogen Synthesis ◽  
Mutant Forms

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6- P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3α and -β genes are replaced with mutant forms (GSK3α/βS21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3α/βS21A/S21A/S9A/S9Amice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6- P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6- P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.

Glycogen Metabolism in Psoriatic Epidermis and in Regenerating Epidermis

1984 ◽  
Vol 67 (3) ◽  
pp. 291-298 ◽  
Author(s):  
C. S. Harmon ◽  
P. J. R. Phizackerley
Keyword(s):  
Glycogen Content ◽  
Glycogen Synthase ◽  
Quantitative Estimation ◽  
Glycogen Synthesis ◽  
Pentose Phosphate ◽  
Normal Epithelium ◽  
Glycogen Synthase Activity ◽  
Psoriatic Lesions ◽  
Wound Epithelium

1. The observation that the glycogen content of epidermis from psoriatic lesions and from regenerating wound epithelium is increased has been confirmed by quantitative estimation. 2. In epidermis from psoriatic lesions, although the proportion of glycogen synthase in the I form is only about 5% of the total and similar to control values, total glycogen synthase activity is increased approximately 4-fold and hence glycogen synthase I activity is increased to the same extent. In contrast, total phosphorylase activity is only slightly increased and, since the proportion of the enzyme in the a form is reduced, phosphorylase a activity is similar to control values. 3. In epidermis from psoriatic lesions, the concentration of UDP-glucose is approximately doubled, and the concentrations of fructose 1,6-bisphosphate and of 6-phosphogluconate are increased approximately 5-fold. It is concluded that rates of glycogen synthesis, of glycolysis and of the pentose phosphate pathway are all enhanced in vivo and in consequence the rate of glucose uptake by psoriatic epidermis must be increased. 4. In the non-involved epidermis of psoriatic patients the glycogen content is within normal limits, and although total glycogen synthase activity is increased the ratio of glycogen synthase I to phosphorylase a is maintained at normal levels by the appropriate phosphorylation of both enzymes. 5. In regenerating wound epithelium in the pig, the changes in enzyme activity and in metabolite concentration closely resemble those found in epithelium from psoriatic lesions except that in wound epithelium the proportion of phosphorylase in the a form is increased relative to normal epithelium.

Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase

1992 ◽  
Vol 70 (7) ◽  
pp. 523-527 ◽  
Author(s):  
Alexander Vardanis
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Glycogen Phosphorylase ◽  
Hydrophobic Interactions ◽  
Glycogen Synthase ◽  
Early Period ◽  
Glycogen Particle ◽  
Molecular Weights ◽  
Glycogen Deposition ◽  
A Minor

The glycogen particle – glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.Key words: glycogen particle, glycogenolysis, glycogenesis, glycogen phosphorylase, glycogen synthase.

scholarly journals Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

2001 ◽  
Vol 2 (3) ◽  
pp. 173-186 ◽  
Author(s):  
Thomas L. Jetton ◽  
Masa Shiota ◽  
Susan M. Knobel ◽  
David W. Piston ◽  
Alan D. Cherrington ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Glycogen Synthesis ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Coupling Mechanism ◽  
Glycogen Accumulation ◽  
Quantitative Image ◽  
Tightly Coupled ◽  
Stimulation Period ◽  
Glycogen Deposition

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.

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