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Biomedical subjects

W Stalmans

Publications and source records attributed to W Stalmans.

At least 91 records · Page 5Linked to original sources
PubMed1987

The use of biochemical parameters for a qualitative and quantitative assessment of ischemic damage to the small-intestinal mucosa.

Lactate dehydrogenase has been measured in the small-intestinal mucosa in order to assess its value as a marker for the effects of ischemia and of reperfusion. The decrease in specific activity of the enzyme illustrates the deleterious effect of reperfusion on the quality of the remaining epithelial cells. However, this parameter fails to detect the loss of epithelial cells, which is the major event during ischemia as well as during reperfusion. In contrast, the expression of enzyme activity per g protein of the underlying intestinal muscle allowed us, in addition, to assess quantitatively the loss of epithelial cells, in good agreement with the histological data.

Animals↗
PubMed1986

Effect of phosphorylation by different protein kinases on the behaviour of glycogen synthase as a substrate for hepatic synthase phosphatases.

Glycogen synthase a from skeletal muscle was phosphorylated in vitro and then used as substrate for the two major synthase phosphatases from liver. Synthase phosphorylated by cAMP-dependent protein kinase (1.4-1.7 P/subunit) was preferentially activated by the cytosolic S-component; in contrast, progressive phosphorylation by casein kinase-1 (0.9-6.5 P/subunit) yielded substrates that were always better dephosphorylated and activated by the glycogen-bound G-component. We have previously isolated from dog liver several types of synthase b that differ by their need for the S- and/or G-component for prompt activation. After additional phosphorylation by a mixture of synthase kinases the activation of these enzyme preparations required the presence of both components.

Animals↗
PubMed1985

An assessment of the importance of intralysosomal and of alpha-amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen-storage disease.

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase. In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of alpha-amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate, already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of alpha-amylase and alpha-glucosidase. Evidence was obtained that gsd hepatocytes are more fragile, and that amylolysis of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick-frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats. The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats. Taken together, the data indicate that only the phosphorolytic degradation of glycogen is metabolically important, and that alpha-amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.

Animals↗
PubMed1985

Calcium ions and glycogen act synergistically as inhibitors of hepatic glycogen-synthase phosphatase.

We investigated the inhibitory effect of Ca2+ in the micromolar range on the activation of glycogen synthase in crude gel-filtered liver extracts [van de Werve (1981) Biochem. Biophys. Res. Commun. 102, 1323-1329]. The magnitude of the inhibition was highly dependent on the glycogen concentration in the final liver extract. Ca2+ inhibited the activation of purified hepatic synthase b by the G-component of synthase phosphatase, as present in the isolated glycogen-protein complex. The cytosolic S-component was not inhibited. Maximal inhibition of the crude G-component occurred at 0.3 microM-Ca2+. The inhibition was not influenced by the addition of either calmodulin or calmodulin antagonists, or by various proteinase inhibitors. The use of purified G-component revealed that the inhibition by 0.3 microM-Ca2+ increased from 45% to 85% when the concentration of glycogen was raised from 1.5 to 20 mg/ml. Muscle glycogen synthase, extensively phosphorylated in vitro, was also used as substrate for purified G-component. Activation and dephosphorylation were similarly inhibited by 0.3 microM-Ca2+, but the magnitude of the inhibition was much greater with the hepatic substrate. No effect of 0.3 microM-Ca2+ was found on the activity of phosphorylase phosphatase in various liver preparations. We conclude that the inhibition of synthase activation by Ca2+ is one of the mechanisms by which cyclic AMP-independent glycogenolytic hormones promote the inactivation of glycogen synthase in the liver, especially in the fed state.

Animals↗
PubMed1984

The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat.

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat-stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor-1 and modulator), did not affect the activity of synthase phosphatase in gel-filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D. Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G-component of synthase phosphatase for efficient conversion to the alpha-form. No G-component could be isolated from livers of adrenalectomized starved rats. Cross-combination of subcellular fractions from control and deficient livers revealed an almost total loss of G-component, with little loss of S-component. This specific loss of functional G-component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan-diabetes.

Adrenal Glands↗
PubMed1984

The hepatic defect in glycogen synthesis in chronic diabetes involves the G-component of synthase phosphatase.

Hepatocytes from normal fed rats and from chronically (90 h) alloxan-diabetic rats were compared. The rate and the extent of activation of glycogen synthase in response to 60 mM-glucose were greatly decreased in diabetes. During incubation of gel-filtered extracts from broken hepatocytes, diabetes only decreased the rate of the activation, which became ultimately complete in either preparation. Synthase phosphatase activity, as measured by the activation of purified hepatic synthase b, was decreased in chronic diabetes. The decrease was proportional to the severity of the diabetes, and reached 90% when the plasma glucose concentration was greater than or equal to 55 mM. In contrast, phosphorylase phosphatase activity was not decreased. Synthase phosphatase activity was progressively restored by treatment with insulin for 20-68 h. During the induction of diabetes and during insulin treatment there was a good correlation between the activity of synthase phosphatase and the maximal activation of synthase in glucose-stimulated hepatocytes from the same livers. The decreased activity of synthase phosphatase in diabetes cannot be explained by an inhibitor. The decrease was much less marked when synthase phosphatase was assayed by the dephosphorylation of 32P-labelled synthase from muscle. This observation suggested a loss of only one component of synthase phosphatase. Cross-combination of subcellular fractions from control rats and from diabetic rats showed a preferential loss of G-component, with little or no loss of S-component. No G-component could be detected in severe diabetes. The concentration of G-component is therefore of critical importance in the glucose-induced activation of glycogen synthase in the liver.

Animals↗
PubMed1983

On the mechanism by which glucocorticoids cause the activation of glycogen synthase in mouse and rat livers.

The administration of glucocorticoids to mice caused within 3 h an inactivation of glycogen phosphorylase and activation of glycogen synthase in their livers. In a Sephadex filtrate of liver extract, as well as in a purified glycogen fraction obtained from treated mice, but not in the same preparations obtained from control mice, glycogen synthase was activated without previous inactivation of phosphorylase. The initial rate of synthase activation in a Sephadex filtrate was proportional to the rate of glycogen synthesis in vivo in the same animal. When the glycogen fraction was isolated in the presence of soluble starch, it could be separated from phosphorylase, phosphorylase phosphatase and synthase phosphatase. When added to a control Sephadex filtrate, this purified glycogen fraction obtained from prednisolone-treated mice relieved synthase phosphatase from inhibition by phosphorylase a, indicating that it contained a transferable 'deinhibiting factor'. This deinhibiting factor appears to be a protein and was further purified by alkyl-Sepharose or DEAE-cellulose chromatography. Another modification introduced by treatment with prednisolone was that phosphorylase phosphatase was 1.5-2-fold more active than in the liver of control mice. This property however did not correlate with the rate of glycogen synthesis in vivo. Administration of actinomycin D prevented the expression of the glucocorticoid effects on the rate of glycogen synthesis in vivo and on the protein phosphatases in vitro. The deinhibition of synthase phosphatase was also observed in isolated rat hepatocytes incubated in the presence of glucocorticoids, but in these preparations synthase was not activated.

Animals↗
PubMed1983

On the mechanism of hepatic glycogenolysis induced by anoxia or cyanide.

Addition of glucagon to isolated hepatocytes increased glycogenolysis and phosphorylase a in a proportional manner. KCN caused slightly more glycogenolysis at considerably lower levels of phosphorylase a; the discrepancy was most pronounced after pretreatment of the hepatocytes with EGTA. When incubated with tagatose, the hepatocytes accumulated tagatose 1-phosphate, a presumed inhibitor of phosphorylase a. In these conditions the glucagon-induced glycogenolysis was blocked, but the glycogen loss caused by KCN or anoxia was not affected. Cyanide and anoxia may allow phosphorylase b and a to become equally active, or they may trigger a non-phosphorolytic glycogenolysis.

Animals↗
PubMed1983

The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat.

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.

Adrenalectomy↗
PubMed1983

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

Animals↗
PubMed1983

Effects of glucose on phosphorylase and glycogen synthase in hepatocytes from diabetic rats.

The effects of glucose on phosphorylase and glycogen synthase were investigated in hepatocytes isolated from acutely (40 h) and chronically (90 h) alloxan-diabetic rats. The glucose-induced inactivation of phosphorylase proceeded normally in all conditions. The ensuing activation of glycogen synthase was slightly blunted in acute diabetes, but became virtually absent in 72 h diabetes of similar severity. In hepatocytes from rats with various degrees of chronic diabetes, the maximal activation of glycogen synthase (at 60 mM-glucose) was inversely correlated with the plasma glucose concentration.

Animals↗
PubMed1982

Induction of hepatic glycogen synthesis by glucocorticoids is not mediated by insulin.

Administration of 0.1 or 1 mg of prednisolone to fed mice caused a 5-fold activation of glycogen synthase in the liver after 3h, without significant changes in the circulating levels of glucose or insulin, or the hepatic concentration of cyclic AMP. Adrenalectomized fasted rats responded to cortisol (10 mg) with an increased glycaemia and a progressive activation of hepatic glycogen synthase after 2-4 h. but without an increase in the very low insulinaemia. These results are incompatible with the prevailing hypothesis that glucocorticoids provoke hepatic glycogen synthesis through an extra secretion of insulin. It is discussed that the acute effect of glucocorticoids is to inhibit rather than stimulate the release of insulin.

Adrenalectomy↗
PubMed1981

The catalytic activity of phosphorylase b in the liver. With a note on the assay in the glycogenolytic direction.

1. The activity and the kinetic properties of purified hepatic phosphorylases a and b from rabbit and rat have been investigated in the glycogenolytic direction with a radiochemical assay. 2. In contrast with the a form, phosphorylase b has an absolute requirement for both AMP and a lyotropic salt. When the latter effectors are included, the b/a-form activity ratio remains low (0.03-0.15) at the hepatic concentration of Pi, because the b form has an exceedingly low affinity for this substrate. 3. Only phosphorylase b is significantly inhibited by glucose, glucose 6-phosphate and MgATP2-. Assays in the presence of substrastes, stimulators and inhibitors in the physiological concentration range indicate that glycogenolysis in the liver depends strictly on the conversion of phosphorylase b into a. Even at 1 mM-AMP the b/a-form activity ratio does not exceed 0.01. 4. Current spectrophotometric procedures for the glycogenolytic assay of phosphorylase in crude liver preparations are highly specific for the a form; the measurement of total phosphorylase (a + b) would require impractical modifications, and is better performed in the direction of glycogen synthesis.

Acetates↗
PubMed1981

Radiochemical methods for the assay of phosphorylase in the direction of glycogenolysis.

Two radiochemical procedures were explored for the determination of phosphorylase activity in the glycogenolytic direction. In the "32P assay method' the formation of labelled glucose 1-phosphate from glycogen and [32P]Pi is measured by the radio-activity that remains soluble after the precipitation of phosphomolybdate with triethylamine. In the "14C assay method' the formation of labelled glucose 1-phosphate from peripherally 14C-labelled glycogen and P1 is determined from the radioactivity that remains soluble after the precipitation of glycogen with ethanol. The 14C assay method requires more preparative work but less circumspection than does the 32P assay method. Both radiochemical methods can be applied where the classical spectrophotometric assay fails. They have the same accuracy and reproducibility, and allow more samples to be handled in parallel. They are not intended for use with crude tissue extracts.

Arsenates↗
PubMed1980

The role of glycogen synthase phosphatase in the glucocorticoid-induced deposition of glycogen in foetal rat liver.

1. The mechanism that underlies the induction of glycogen synthesis in the foetal rat liver by glucocorticoids was reinvestigated in conditions where the accumulation of glycogen is either precociously induced with dexamethasone or inhibited by steroid deprivation. It appears that glucocorticoids act as the physiological trigger for glycogen synthesis by inducing both glycogen synthase (a known effect) and its activating enzyme, glycogen synthase phosphatase. 2. The activity of glycogen synthase phosphatase in adult liver stems from the interaction of two protein components [Doperé, Vanstapel & Stalmans (1980) Eur. J. Biochem. 104, 137--146]. Two independent experimental approaches indicate that the cytosolic 'S-component' is already well developed in the foetal liver before the onset of glycogen synthesis. The manifold glucocorticoid-dependent increase in synthase phosphatase activity during late gestation must be attributed to the specific development of the glycogen-bound 'G-component'.

Animals↗
PubMed1980

Glycogen-synthase phosphatase activity in rat liver. Two protein components and their requirement for the activation of different types of substrate.

Three subfractions of glycogen synthase b (termed b1, b2, b3) have been isolated from the glycogen fraction of dog liver on the basis of a different affinity for DEAE-cellulose. Their kinetic properties and chromatographic behaviour are compatible with the presence of an increasing number of phosphorylated sites from synthase b1 towards b3. Synthase phosphatase activity in rat liver stems from two heat-labile and trypsin-labile proteins. These components are conveniently prepared from the cytosolic fraction of glycogen-depleted liver; the 'G-component' of the phosphatase co-sediments with added particulate glycogen, whereas the 'S-component' remains in the supernatant. The G-component alone did not convert any available synthase b to the a form. The synthase phosphatase activity of the S-component was variable according to the actual type of substrate. When acting on synthase b2 and b3, the S-component had a low phosphatase activity that was increased 7-fold and 11-fold, respectively, upon addition of the G-component. Synthase b1, however, was efficiently activated by the S-component, and only 35% faster in the presence of both components. When the cytosolic fraction of glycogen-depleted livers was analysed by sucrose-gradient centrifugation a single peak of phosphatase activity (S20, W = 10.2 S; provisional Mr = 254000) was detected with synthase b2 as substrate. In addition to this peak, presumably an S-G complex, synthase b1 also identified free S-component of lower and heterogeneous molecular weight. Our results illustrate in general the influence of the type of synthase b on the detection of synthase phosphatase activity, and specifically may provide an explanation for some discrepant reports on the subcellular distribution of the enzyme.

Animals↗