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1.
This study, using 13C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from 13C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10-8 mol·1-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.  相似文献   

2.
The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE+ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min-1·g wet weight-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l-1 for glycogen phosphorylase a and 23.6 mmol·l-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K a of 0.176±0.004 mmol·l-1. Michaelis constant and K a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I 50 concentration of inhibitor that reduces enzymes velocity by 50% - K a concentration of activator that produces half-maximal activation of enzyme activity - K m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - Pi morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V max enzyme maximal velocity  相似文献   

3.
Active and total phosphorylase activity, using labelled C14-glucose-1-phosphate as the substrate, is demonstrated by histoautoradiographic method. This method can demonstrate the polysaccharide synthesizedin vitro by phosphorylase without intervention from the unlabelled pre-existing glycogen. C14-glucose can not replace C14-glucose-1-phosphate as substrate. The distribution of phosphorylase in tissue sections, except in cases of very low activity, is similar to that obtained by customary dilute Lugol's iodine staining method. The relative difference of intensity between active and total phosphorylase, as revealed by iodine staining, is also reflected by histoautoradiographic method. Histoautoradiographic method has several advantages over the iodine staining method. This method is more sensitive for demonstration of very low phosphorylase activity which may escape detection by iodine staining. Branching enzyme activity, especially when it favors synthesis of glycogen type of polysaccharide instead of amylopectin type, can be better detected by this method. Active phosphorylase substrate medium can be used to demonstrate this activity in plant tissues, where the presence of pre-existing starch often prohibits the use of iodine staining method. Stripping film method for autoradiography is recommended for the study of this enzyme activity.  相似文献   

4.
Summary Adult Manduca sexta feed very irregularly in the laboratory, and many adult males never feed. Feeding adults live longer and feeding females lay many more eggs; however, in both feeding (sugar water) and starving adults a decrease of metabolic reserves is observed. Carbohydrates disappear from hemolymph and from fat body. Fat body lipid also decreases, while hemolymph lipid concentration increases strongly in starving adults. The activity of fat body glycogen phosphorylase increases strongly in starving adult M. sexta. The activity of glycogen phosphorylase is correlated inversely with hemolymph sugar concentration. Injected trehalose inactivates glycogen phosphorylase within 2 h, and lowers the hemolymph lipid level within 6 h. In starving adult M. sexta, neither the activation of glycogen phosphorylase nor the increase of hemolymph lipid concentration depends on adipokinetic hormone, since cardiacectomy does not prevent the activation of glycogen phosphorylase nor the increase of hemolymph lipid level.Abbreviations AKH adipokinetic hormone - EDTA ethylenediamine tetraacetate Present address: Department of Biochemistry and Center for Insect Science, The University of Arizona, Tucson, AZ 85721, USA  相似文献   

5.
The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R2 = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.  相似文献   

6.
Recently, oleanolic acid was found to be an inhibitor of glycogen phosphorylase. For further structural modification, we have synthesized several dimers of oleanolic acid by using amide, ester, or triazole linkage with click chemistry. The click chemistry was shown to be the most efficient method for the dimer synthesis. Nearly quantitative yield of triazole‐linked dimers was obtained. Biological evaluation of the synthesized dimers as inhibitors of glycogen phosphorylase has been described. Four of six dimers exhibited inhibitory activity against rabbit muscle glycogen phosphorylase a (RMGPa), with compounds 2 and 7 as the most potent inhibitors, which displayed an IC50 value (ca. 3 μM ) lower than that of oleanolic acid (IC50=14 μM ).  相似文献   

7.
Activation and inactivation of fat body glycogen phosphorylase was investigated in ligated abdomens of larval Manduca sexta and in vitro. After maximal activation through Manduca adipokinetic hormone (AKH) or chilling, inactivation of glycogen phosphorylase commenced as soon as the stimulus for the activation was removed indicating that the enzyme system in the fat body is fine-tuned to low phosphorylase activities which is necessary to allow glycogen synthesis. In intact ligated abdomens phosphorylase can be activated repeatedly by either stimulus showing that the fat body system does not lose its responsiveness. It was impossible to achieve complete conversion of the inactive form of phosphorylase into the active form even after administration of AKH and simultaneous chilling. © 1992 Wiley-Liss, Inc.  相似文献   

8.
Johannes Lehmann 《Planta》1973,114(1):51-61
Summary Tests for enzymes of gluconeogenesis and of the synthesis and degradation of sucrose and polysaccharides have been carried out in the phloem exudate of Cucurbita pepo. All the enzymes which are necessary for the synthesis of sucrose and polysaccharides from metabolites of the citric acid cycle were found to be present in the exudate, except phosphoenolpyruvate carboxykinase. The polysaccharide synthetase was found to exhibit higher activity with glycogen (which is an unnatural polysaccharide in higher plants) than with starch. In addition, polysaccharide synthetase activity could be increased remarkably with 2 mM glucose-6-phosphate and glycogen as primer. Among the enzymes which catabolize sucrose and polysaccharides (phosphorylase, invertase, sucrose phosphorylase), only sucrose phosphorylase showed activity.  相似文献   

9.
Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.  相似文献   

10.
This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5"-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5"-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.  相似文献   

11.
12.
Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in Vmax, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.  相似文献   

13.
Summary We have recently demonstrated that the activity of liver glycogen phosphorylase, the rate-limiting enzyme of glycogenolysis, is elevated in genetically diabetic (db/db) mouse and that it is primarily due to the presence of increased amounts of this enzyme. In the present study, we examined the turnover of glycogen phosphorylase in vivo in order to elucidate the mechanism for this specific increase. The rate of phosphorylase synthesis was slightly decreased in the diabetic mouse compared to controls. However, the relative rates of synthesis were similar in these two groups. The rate of degradation of this enzyme was decreased 20% (p<0.05) in the diabetic mouse compared to controls. More importantly, the relative rate of degradation of phosphorylase was found to be lower in the diabetic animals. This indicates that the elevated concentration of phosphorylase in the liver of the db/db mouse is likely due to a specific decrease in its rate of degradation.  相似文献   

14.
Mantle tissue pieces from adult Otala lactea continuously synthesized glycogen over a 72-h incubation period. Acid-saline extract of the cerebral ganglia inhibited glycogen synthesis by mantle tissue in vitro. This effect was dose-dependent. The glycogen reduction factor from the cerebral ganglia was heat stable, protease sensitive, and relatively hydrophobic. The cerebral ganglia extract also stimulated mantle glycogen phosphorylase in vitro in a dose-dependent manner. The results suggest the presence of a hyperglycemic factor in the cerebral ganglia of Otala. The molecular weight of this factor, estimated by size-exclusion chromatography, was approximately 10,000. Mammalian glucagon had no significant effect on glycogen synthesis by the mantle pieces. Accepted: 17 January 2000  相似文献   

15.
Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1–GL) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1–GL complex, suppressing glycogen synthesis. Consequently, the interaction of GL with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the GL–phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the GL protein. The resulting GLY284F/Y284F mice display hepatic PP1–GL activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of GL by phosphorylase a operates in vivo. GLY284F/Y284F and GLY284F/+ mice display improved glucose tolerance compared with GL+/+ littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the GL–phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the GLY284F/Y284F mice lose more body weight and display decreased blood glucose levels in comparison with their GL+/+ littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of GL may prevent futile glucose–glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1–GL by phosphorylase a in mammals.  相似文献   

16.
The influence of starvation on carbohydrate metabolism in fifth instar larvae of Manduca sexta was studied. The percentage of active fat body glycogen phosphorylase increased from 10% to approximately 50% within 3 h of starvation; afterward the enzyme was slowly inactivated. The increase of phosphorylase activity might have been caused by a peptide(s) from the CC. The amount of fat body glycogen in starved animals decreased over 24 h by approximately 20 mg. The released glucose molecules seem to be converted mainly to trehalose because the hemolymph trehalose concentration in starved animals was always slightly higher than in the fed controls, and the glucose concentration decreased even when phosphorylase was activated. The chitosan content in starved larvae increased during the first 9 h of treatment to the same extent as in fed controls. It is suggested that fat body glycogen phosphorylase was activated during starvation to provide substrates for chitin synthesis and energy metabolism.  相似文献   

17.
The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca2+, 10 mm Mg2+, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca2+-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [32P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [32P]-phosphorylase.  相似文献   

18.
Glycogen phosphorylase in Tetrahymena pyriformis was activated by a Mg2+ ATP-dependent process and this activation was further increased by the addition of cyclic AMP. When the enzyme activity in subcellular fractions was measured, it was largely associated with the glycogen fraction but was no longer activated by ATP and cyclic AMP. Mixing the glycogen fraction and cytosol fraction together restored the effects of ATP and cyclic AMP on phosphorylase activity. These findings suggest that glycogen phosphorylase associated with Tetrahymena glycogen granules may be regulated by cytosolic factor(s) with cyclic AMP.  相似文献   

19.
We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.  相似文献   

20.
The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity,an intermediate plateau,and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min–1;1 M GuHCl, 25°C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.  相似文献   

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