Glycogen synthesis by the direct or indirect pathways depends on glucose availability: In vivo studies in frog oocytes (original) (raw)
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Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway
The Biochemical journal, 1996
Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor-product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H, U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but signifi...
Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions
Archives of Biochemistry and Biophysics, 2003
Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K m app for UDP-glucose in vivo was 1.4 mM (in the presence or absence of glucose-6-P). The in vitro value was 6 mM and was reduced by glucose-6-P to 0.8 mM. Under both conditions (in vitro and in vivo) V max was 0.2 mUnits per oocyte in the absence of glucose-6-P. V max in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 lM and, depending on substrate concentrations, 50-100 lM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.
FEBS Letters, 2013
Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and À0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.
Frog Oocytes: A Living Test Tube for Studies on Metabolic Regulation
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2001
This review is intended to illustrate how live frog oocytes may be advantageously used to address the study of some problems of in vivo glucose metabolism. Glucose microinjected into the cells is preferentially committed to glycogen synthesis. We present evidence showing that both the direct and indirect pathways for polysaccharide deposition are operative in oocytes. A small amount of the injected glucose (<5%) is released as labeled CO 2 mainly through the pentose-P pathway. Coinjection of NADP C and glucose signi cantly stimulates 14 CO 2 production, half-maximal stimulation being obtained at 0.13 mM. Finally, we show the use of frog oocytes to measure in vivo the control coef cient of hexokinase on glycogen synthesis and the pentose-P pathway. A value of 0.7 was found for the control coef cient of hexokinase on glycogen synthesis, while the enzyme has no control at all over the pentose-P pathway. Therefore, the frog oocyte may be used as a living test tube for the study of almost any metabolic process of interest.
Glycolysis is operative in amphibian oocytes
FEBS Letters, 1994
It is generally accepted that in frog full-grown oocytes glycolysis is absent and that carbon metabolic flux is largely directed to glycogen synthesis. Use of an anion exchange pellicular resin for analytical resolution of intermediates in perchloric acid extracts of oocytes has allowed us to observe the formation of labelled lactate after microinjection of [u-i4C]glucose. Further, formation of ["P]ATP was observed after microinjection of 32P-labelled glucose-6-P, fructose-6-P or fructose-1,6-b&P, either in the presence or absence of 0.1 mM cyanide. The presence of phosphofructokinase activity, previously thought to be absent in oocytes, is also reported. These findings indicate that glycolysis to lactate is operative in frog oocytes.
Archives of Biochemistry and Biophysics, 1997
Glycogen is the main product of glucose metabolism Glycogen synthesis following glucose microinjection in fully grown amphibian oocytes (1, 2). Recently, we in frog oocytes proceeds preferentially by an indirect have reported that in frog oocytes, glycogen synthesis pathway involving gluconeogenesis from triose comafter labeled glucose microinjection occurs preferenpounds. Because of the known regulatory role of fructially by an ''indirect'' pathway (3) that involves glycolytose-2,6-bisP on glucose utilization in most vertebrate sis to three-carbon compounds which are then used by tissues we coinjected [U-14 C]glucose and fructose-2,6gluconeogenesis for the resynthesis of hexoses-P and bisP into oocytes and observed a marked inhibition of UDP-glucose, the proximate glycogen precursors. Such label incorporation into glycogen, with an I 50 value of an alternative pathway was reported a few years ago 2 mM, which is similar to the value measured for the in for mammalian liver (for reviews see 4-7), isolated hevitro inhibition of oocyte fructose-1,6-bisphosphatase. patocytes (8-10), and cultured astrocytes (11). The key Other hexoses-bisP were tested: 2,5-anhydromannitolgluconeogenic enzymatic activities, phosphoenolpyr-1,6-bisP was as effective as inhibitor as fructose-2,6uvate-carboxykinase and fructose-1,6-bisphosphatase bisP; glucose-1,6-bisP showed some effect although (FBPase), 3 are present in frog oocytes (3, 12). Oocyte 50% inhibition was obtained at a concentration 10 FBPase, as well as many other bisphosphatases, is times higher than with fructose-2,6-bisP; fructose-1,6strongly inhibited by fructose-2,6-bisP. Therefore, fruc-bisP had no effect at all. The inhibition pattern for tose-2,6-bisP could play an important role in the reguthe in vivo glycogen synthesis by these analogs closely lation of glucose utilization commited for glycogen synmatched the one obtained with partially purified oothesis through the indirect gluconeogenic pathway. cyte fructose-1,6-bisphosphatase. The intracellular In this paper we report the effect of microinjected concentration of fructose-2,6-bisP in unperturbed oofructose-2,6-bisP on glycogen synthesis in vivo and the cytes was found to be between 0.1 and 0.2 mM. Fruccellular concentration of the metabolite, together with tose-6-phosphate,2-kinase levels measured in oocyte fructose-6-phosphate,2-kinase activity. We have found homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP mi-that fructose-2,6-bisP does indeed control glycogen syncroinjected into the oocytes was almost completely de-thesis via the indirect pathway mainly through its acgraded, suggesting that fructose-2,6-bisphosphatase is tion upon oocyte FBPase. The physiological significance active in vivo. The results presented in this paper indiof these findings will be discussed. cate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-MATERIALS AND METHODS grown oocytes.
Anomeric specificity of d-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms
Biochimie, 2004
The anomeric specificity of D-[U-14 C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37°C and then incubated for 5 min at the same temperature in the presence of aor b-D-[U-14 C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D 2 O/H 2 O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with a-D-glucose than with b-D-glucose suggesting a higher rate of b-D-glucose than a-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the a/b ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for a-D-glucose.
BBA Clinical, 2016
In the human body, glycogen is a branched polymer of glucose stored mainly in the liver and the skeletal muscle that supplies glucose to the blood stream during fasting periods and to the muscle cells during muscle contraction. Glycogen has been identified in other tissues such as brain, heart, kidney, adipose tissue, and erythrocytes, but glycogen function in these tissues is mostly unknown. Glycogen synthesis requires a series of reactions that include glucose entrance into the cell through transporters, phosphorylation of glucose to glucose 6-phosphate, isomerization to glucose 1-phosphate, and formation of uridine 5ʹ-diphosphate-glucose, which is the direct glucose donor for glycogen synthesis. Glycogenin catalyzes the formation of a short glucose polymer that is extended by the action of glycogen synthase. Glycogen branching enzyme introduces branch points in the glycogen particle at even intervals. Laforin and malin are proteins involved in glycogen assembly but their specific function remains elusive in humans. Glycogen is accumulated in the liver primarily during the postprandial period and in the skeletal muscle predominantly after exercise. In the cytosol, glycogen breakdown or glycogenolysis is carried out by two enzymes, glycogen phosphorylase which releases glucose 1-phosphate from the linear chains of glycogen, and glycogen debranching enzyme which untangles the branch points. In the lysosomes, glycogen degradation is catalyzed by α-glucosidase. The glucose 6-phosphatase system catalyzes the dephosphorylation of glucose 6-phosphate to glucose, a necessary step for free glucose to leave the cell. Mutations in the genes encoding the enzymes involved in glycogen metabolism cause glycogen storage diseases.
Control of glycogen deposition
FEBS Letters, 2003
Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more e¡ective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit speci¢c metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.