Biomedical subjects

W Stalmans

Publications and source records attributed to W Stalmans.

At least 73 records · Page 4Linked to original sources

PubMed1990

The interaction of phosphorylase a with D-glucose displays alpha-stereospecificity.

Half-maximal inhibition of phosphorylase a required a much lower concentration of alpha-D-glucose (4 mM) than of the beta-anomer (14 mM) and of 1-deoxyglucose (about 25 mM). beta-D-Glucose was almost ineffective at concentrations of 1-2.5 mM, but at 50 mM the two anomers were equipotent. A similar picture emerged when the stimulatory effects of the glucose anomers and of 1-deoxyglucose were investigated on the inactivation of phosphorylase by phosphorylase phosphatase. However, upon addition of either glucose anomer (5-20 mM) to a suspension of isolated hepatocytes, the inactivation of phosphorylase occurred at the same rate. It is shown that, in the latter conditions, the rate of intracellular mutarotation considerably exceeds the rate of glucose transport. This results presumably in a rapid anomeric equilibrium in the liver cells.

Animals↗

PubMed1990

Oral administration of vanadate to streptozotocin-diabetic rats restores the glucose-induced activation of liver glycogen synthase.

Isolated hepatocytes from streptozotocin-diabetic rats failed to respond to a glucose load with an activation of glycogen synthase. This lesion was associated with severely decreased activities of glycogen-synthase phosphatase and of glucokinase. All these defects were abolished after consumption for 13-18 days of drinking water containing Na3VO4 (0.7 mg/ml), and they were partially restored after 3.5 days, when the blood glucose concentration was already normalized. In all conditions the maximal extent of activation of glycogen synthase in cells closely parallelled the activity of glycogen-synthase phosphatase.

Animals↗

PubMed1990

The cytosolic concentration of phosphate determines the maximal rate of glycogenolysis in perfused rat liver.

Glycogenolysis was studied in glycogen-rich perfused livers in which glycogen phosphorylase was fully converted into the a form by exposure of the livers to dibutyryl cyclic AMP. We monitored intracellular Pi by 31P n.m.r. Perfusion with Pi-free medium during 30 min caused a progressive decrease of the Pi signal to 50% of its initial value. In contrast, exposure of the livers to KCN and/or 2,4-dinitrophenol resulted in a rapid doubling of the Pi signal. Alterations in the intracellular Pi coincided with proportional changes in the rate of hepatic glycogenolysis (measured as the output of glucose plus lactate). The results indicate that the rate of glycogenolysis catalysed by phosphorylase a depends linearly on the hepatic Pi concentration. Hence the Km of phosphorylase a for its substrate Pi must be considerably higher than the concentrations that occur in the cytosol, even during hypoxia.

2,4-Dinitrophenol↗

PubMed1990

Influences of ischemia and reperfusion on the feline small-intestinal mucosa.

The alterations of several small-intestinal mucosal enzymes have been examined in cats that underwent different periods (1-4 hr) of occlusion of the superior mesenteric artery, followed by 4 hr of reperfusion. The damage progressed during ischemia and reperfusion from the villus tips to the crypts: first, there was a rapid decrease in the activity of maltase, a brush-border enzyme; a slower decline occurred in two cytoplasmic enzymes, aldolase A (with preferential location in feline villus cells) and lactate dehydrogenase (with an ubiquitous distribution); a lag preceded the decrease in aldolase B (a cytoplasmic enzyme shown to occur mainly in feline crypt cells). For all these enzymes, the initial period of reperfusion was associated with a greater decrease in enzyme activity than persisting ischemia. By determination of the unsedimentable proportion of glutamate dehydrogenase (a mitochondrial matrix enzyme) and of acid phosphatase (a lysosomal enzyme) it was demonstrated that ischemia caused important mitochondrial damage before the cells were lost, whereas no lysosomal damage was observed in any condition. These sensitive parameters of cell damage can serve as a criterion for an adequate evaluation of potential cytoprotective agents.

Acid Phosphatase↗

PubMed1990

Short-term hormonal control of protein phosphatases involved in hepatic glycogen metabolism.

The prominent protein phosphatases involved in liver glycogen metabolism are the AMD (ATP, Mg-dependent, type-1) and PCS (polycation-stimulated, type-2A) phosphatases. The glycogen synthase phosphatase activity, measured from the rate of activation of liver glycogen synthase, is virtually accounted for by AMD phosphatases; the bulk of the activity belongs to the glycogen-bound protein phosphatase G and a small part is present in the cytosol. The major part of the phosphorylase phosphatase activity present in the post-mitochondrial supernatant is shared by protein phosphatase G and cytosolic enzymes, and a minor part belongs to a microsomal AMD phosphatase. In the liver cytosol, the phosphorylase phosphatase activity is about equally distributed between AMD and PCS phosphatases. Studies in vivo as well as on isolated, perfused livers have shown that glucagon (which raises the level of cyclic AMP) as well as vasopressin (which increases the cytosolic Ca2+ concentration) decrease the phosphorylase phosphatase activity in liver extract or cytosol (filtered through Sephadex G-25) by about 25% within a few minutes. These effects were not additive, and the activity of glycogen synthase phosphatase was not affected. Conversely, insulin as well as glucose increased both phosphatase activities by about 25%, and these effects were additive. Vanadate mimicked the effect of insulin on the perfused liver. All the activity changes were only observed when the assays were performed at high tissue concentration. Upon subcellular fractionation all the effects were well expressed in the cytosol, but not in the particulate fraction (glycogen and microsomes). However, quantitatively the hormonal responses were largely lost during the fractionation procedure; they could be restored by recombination of the liver cytosol from a hormone-treated rat with the particulate fraction from either a treated or an untreated animal. It appears that the effects of glucagon, insulin and glucose are mediated by cytosolic, transferable effectors of the Vmax of protein phosphatases. These effectors are eluted in the void volume of a Sephadex G-25 column. Rats of the gsd/gsd strain, which have a genetic deficiency of hepatic phosphorylase kinase, responded to an injection of insulin plus glucose with a normal increase in the cytosolic phosphorylase phosphatase activity. In contrast, they failed to respond to glucagon as well as vasopressin. A transient 80% inhibition of the phosphorylase phosphatase activity could be induced in vitro in a concentrate liver cytosol from Wistar rats upon addition of MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Decreased activity and impaired hormonal control of protein phosphatases in rat livers with a deficiency of phosphorylase kinase.

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.

Animals↗

PubMed1989

The antiglycogenolytic action of 1-deoxynojirimycin results from a specific inhibition of the alpha-1,6-glucosidase activity of the debranching enzyme.

The glucose analogue 1-deoxynojirimycin (dNOJ) and some of its N-substituted derivatives have recently been described as potent inhibitors of the hepatic glycogenolysis induced by glucagon, Ca2+ ionophores or anoxia. The inhibition increased with time, in spite of a persistently high level of phosphorylase a [Bollen, M., Vandebroeck, A. & Stalmans, W. (1988) Biochem. Pharmacol. 37, 905-909]. dNOJ equilibrates within 1 min across the plasma membrane of hepatocytes. It is not phosphorylated or oxidized in the cell. The observation that dNOJ did not affect gluconeogenesis excludes the possibility that glucose-6-phosphatase is the target for the inhibition of glucose production from glycogen. Neither were the catalytic activities of phosphoglucomutase and phosphorylase a affected by the compound. dNOJ and two N-substituted derivatives inhibited instantaneously and completely the alpha-1,6-glucosidase activity of the debranching enzyme, with I50 values in the mumolar range. In contrast, the glucanotransferase activity of the latter enzyme was not inhibited by the compounds at 0.2 mM. The effect of dNOJ was further studied in an in vitro model system of glycogenolysis. The results were compatible with a block of glycogenolysis at the time when phosphorylase has removed the available glucosyl residues from the outer chains of the glycogen particles. This mechanism appears to account for the lag in the response of glycogenolysis to dNOJ.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1989

[Deficiency in hepatic uptake of glucose in chronic diabetes mellitus].

In insulin-dependent diabetes mellitus there is a deficient post-prandial uptake of glucose and storage as glycogen in the liver. This impairment is due to an intrinsic hepatic defect that has been investigated with the use of isolated liver cells. Glycogen synthase catalyzes the rate-limiting step in the synthesis of glycogen. In response to an increased glucose concentration, this enzyme is activated in normal hepatocytes through dephosphorylation of seryl residues by a glycogen-bound "protein phosphatase G". Hepatocytes isolated from alloxan diabetes rats have lost the ability to activate glycogen synthase in response to an increased glucose concentration. The magnitude of the latter defect corresponds to the severity of the diabetes, as judged from the level of glycaemia. The defect is explained by an impaired function of protein phosphatase G. The latter enzyme consists of a catalytic subunit (37 kDa) associated with a large glycogen-binding subunit (161 kDa) and other regulatory polypeptides. It appears that in diabetes an essential regulatory subunit is deficient. Studies in animals with distinct types of spontaneous diabetes revealed that lack of insulin, rather than chronic hyperglycaemia, explains the deficient activity of protein phosphatase G.

Animals↗

PubMed1988

The hepatic glycogenolysis induced by reversible ischaemia or KCN is exclusively catalysed by phosphorylase a.

1. Ischaemia was applied for 30 min to the liver of Wistar rats and of gsd/gsd rats, which have a genetic deficiency of phosphorylase kinase. The rate of glycogenolysis corresponded closely to the concentration of phosphorylase a. The loss of glycogen from Wistar livers was accounted for by the intrahepatic increase in glucose plus lactate. Further, the accumulation of oligosaccharides was negligible in the gsd/gsd liver. 2. Isolated hepatocytes from Wistar and gsd/gsd rats were incubated for 40 min in the presence of either KCN or glucagon. Again, the production of glucose plus lactate was strictly dependent on the presence of phosphorylase a. However, the catalytic efficiency of phosphorylase a was about 2-fold higher in the presence of KCN. 3. We conclude that the hepatic glycogenolysis induced by anoxia and by KCN is solely mediated by phosphorylase a. The higher catalytic activity of phosphorylase a under these circumstances could be due to an increased concentration of the substrate Pi.

Animals↗

PubMed1988

Acute regulation of hepatic protein phosphatases by glucagon, insulin, and glucose.

The intravenous administration of glucagon to anesthetized rats resulted within 5 min in a 20% drop in the hepatic phosphorylase phosphatase activity, as measured in a post-mitochondrial supernatant at low dilution, but it did not affect the activity of glycogensynthase phosphatase. On the other hand, the injection of insulin plus glucose caused increases by about 35% in both phosphatase activities. Upon subcellular fractionation these effects were recovered in the cytosol, but not in the glycogen/microsomal fraction. However, activity changes in the latter fraction were observed after recombination with the liver cytosol from a hormone-treated animal. Preincubation of the liver cytosol with modulator protein (a specific inhibitor of type-1 protein phosphatases) cancelled the activity changes induced by insulin plus glucose. No hormonal effects on hepatic protein phosphatase activities were observed when the fractions were either diluted an additional 10-fold or pretreated with trypsin. An acute hormonal regulation of protein phosphatases could also be demonstrated in the perfused liver. When added to the perfusion medium, glucose as well as insulin increased the cytosolic protein phosphatase activities by about 25%. Their effect was additive, irrespective of the order of addition. On the other hand, the addition of glucagon and/or vasopressin resulted in a 20% drop in the phosphorylase phosphatase activity. The presence of glucagon did not interfere with the effectiveness of insulin, and vice versa. The changes in the phosphorylase phosphatase activities induced by glucagon, insulin, and glucose represented changes in the Vmax only. We propose that the acute control of the hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities is mediated by transferable, cytosolic effector(s).

Animals↗

PubMed1988

Fluorine compounds inhibit the conversion of active type-1 protein phosphatases into the ATPMg-dependent form.

1. The modulator protein slowly converts the glycogen-bound protein phosphatase from liver, as well as its catalytic subunit, into an inactive form that requires protein kinase FA and MgATP for reactivation. The inactivation process could be completely prevented by addition of either 0.3 mM-NaF or 0.3 mM-phenylmethanesulphonyl fluoride (PMSF). The effectiveness of the proteinase inhibitor was not due to production of free fluoride. With the catalytic subunit a half-maximal effect of either fluorine compound was obtained at 25-50 microM. 2. The inactivation process was instantaneously blocked by the addition of NaF or PMSF at any moment during the incubation of the catalytic subunit with modulator. This fluoride effect was reversible. It did not result from a decreased affinity of modulator for the catalytic subunit. The use of analogues of PMSF showed that the fluorine atom was essential, but structural aspects were also an important determinant. 3. The relative efficiency of fluorine compounds in preventing the inactivation of the catalytic subunit by modulator corresponded to their relative potency as inhibitors of the phosphorylase phosphatase activity, but the latter effect required at least 20-fold higher effector concentrations. Incubation of the catalytic subunit with 10 mM-PMSF or -NaF caused an irreversible inhibition of the enzyme. 4. It is possible to prepare stable complexes of catalytic subunit and modulator, either active or ATPMg-dependent. Both species displayed the same molecular size during gel filtration. The inactive complex could be reactivated by incubation with MgATP and protein kinase FA. NaF and PMSF increased the final extent of re-activation at limiting concentrations of the protein kinase.

Adenosine Triphosphate↗

PubMed1988

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions.

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.

Animals↗

PubMed1988

The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen.

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).

Animals↗

PubMed1988

1-Deoxynojirimycin and related compounds inhibit glycogenolysis in the liver without affecting the concentration of phosphorylase a.

Administration in vivo of the alpha-glucosidase inhibitors 1-deoxynojirimycin and its derivatives BAY m 1099 (miglitol) and BAY o 1248 resulted in a dose- and time-dependent decrease in the rate of hepatic glycogenolysis induced by glucagon. This represents a direct effect on the liver, since it could be reproduced on isolated hepatocytes. The amount of glucose produced by hepatocytes over a period of 10-20 min after addition of glucagon was decreased by about 70, 60 and 45% in the presence of maximally effective concentrations of BAY o 1248, deoxynojirimycin, and BAY m 1099, respectively. Half-maximal effects were observed at inhibitor concentrations between 20 and 100 microM. The concentrations of phosphorylase a and glycogen synthase a were not affected by inclusion of the alpha-glucosidase inhibitors in the hepatocyte suspensions. Thus, the antiglycogenolytic action of these compounds is not mediated by an altered activation state of the rate-limiting enzymes of glycogenolysis and of glycogen synthesis.

1-Deoxynojirimycin↗

PubMed1988

Differences in liver glycogen-synthase phosphatase activity in rodents with spontaneous insulin-dependent and non-insulin-dependent diabetes.

The activation of glycogen synthase after addition of glucose to isolated hepatocytes became impaired in BB rats after the development of insulin-dependent diabetes. This defect was associated with a decreased hepatic synthase phosphatase activity. Both features correspond closely to previous observations on alloxan-diabetic rats. In contrast, in hyperinsulinaemic db/db mice with a similarly increased plasma glucose concentration (non-insulin-dependent diabetes), the synthase phosphatase activity was essentially normal. We conclude that the decreased hepatic synthase phosphatase activity in insulin-dependent diabetes in rodents is due to the lack of insulin, rather than to the increased intrahepatic glucose concentration.

Animals↗

PubMed1988

The effect of the thyroid status on the activation of glycogen synthase in liver cells.

Isolated hepatocytes from hyperthyroid and euthyroid rats showed the same rate and extent of activation of glycogen synthase after addition of glucose (10 mM or 60 mM). In liver cells from hypothyroid rats this activation occurred at a 7-fold lower rate. However, complete activation of glycogen synthase occurred eventually in broken-cell preparations from either source during incubation in vitro. Glycogen synthase phosphatase was then quantitatively assayed in liver homogenates with exogenous synthase b as substrate. These assays revealed an increased synthase phosphatase activity (approximately 160%) in the hyperthyroid liver and a decreased activity (to approximately 60%) in the livers from hypothyroid rats. These activity changes involved both the cytosolic and the glycogen-bound synthase phosphatase. The increase in the activity of synthase phosphatase after the administration of T3 became maximal after 48 h. We conclude that thyroid hormones control hepatic glycogen synthesis, at least partly by an effect on synthase phosphatase.

Animals↗

PubMed1987

High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a.

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.

Animals↗

PubMed1987

Control of glycogen synthesis in health and disease.

Investigations in our laboratory have shown that the activity of glycogen synthase phosphatase in the liver is shared by at least two functionally distinct proteins: a G-component, which is tightly associated with glycogen particles, and a soluble S-component. Most preparations of glycogen synthase-b that are isolated from the liver of fed glucagon-treated animals require the presence of both components in order to be converted to synthase-a. The G-component is subject to control mechanisms that do not affect the S-component. Its activity is strongly inhibited by phosphorylase-a. This feature explains why glycogen synthesis and glycogenolysis do not normally occur simultaneously, except in the glycogen-depleted liver, where a futile cycle may occur. Experiments in vitro have shown that a minimal glycogen concentration is required to ensure the interaction between the G-component and phosphorylase-a. The G-component is also selectively inhibited by Ca2+, and the magnitude of this inhibition depends markedly on the glycogen concentration. The latter inhibition is probably one of the mechanisms by which cyclic adenosine monophosphate (cAMP)-independent glycogenolytic agents achieve the inactivation of glycogen synthase in the liver. Glucocorticoid hormones and insulin are required for the induction and/or maintenance of the G-component in the liver. During the development of the fetal rat, glucocorticoids induce the G-component in the liver. This is an essential event in the glucocorticoid-triggered deposition of glycogen in the fetal liver. A functional adrenal cortex is also required in the adult animal to prevent a loss of the capacity for hepatic glycogen storage during starvation. The latter capacity depends on the concentration of functional G-component in the liver. Chronic diabetes causes a similar functional loss. However, the effect of glucocorticoids is not mediated by a putative secretion of insulin.

Animals↗