A regulatory role for cortisol in muscle glycogen metabolism in rainbow trout Oncorhynchus mykiss Walbaum.

To test the hypothesis that cortisol has a regulatory role in fish muscle glycogenesis post-exercise, rainbow trout were treated 1 h prior to exercise with either saline (control) or metyrapone (2-methyl-1, 2-di-3-pyridyl-1-propanone) to block cortisol synthesis. Following exercise (time 0), half of the metyrapone-treated fish received a single injection of cortisol, to mimic the post-exercise rise usually observed. Muscle glycogen and the relative activities of glycogen phosphorylase a (Phos a) and glycogen synthase I (GSase I), regulatory enzymes for glycogen resynthesis, were monitored 4 h post-exercise. Metyrapone treatment succeeded in blocking the post-exercise rise in plasma cortisol (17+/-2 vs 118+/-13 ng ml(-1) in controls at time 0), and cortisol injection resulted in a larger and more prolonged cortisol increase than in controls (159+/-22 vs 121+/-14 ng ml(-1) in controls at 1 h). Muscle glycogen was completely restored in the metyrapone-treated fish within 2 h after exercise (8.3+/-0.6 vs 8+/-0.7 micromol g(-1) pre-exercise), only partially restored in control fish at 4 h (5.4+/-01.4 vs 8.8+/-1.3 micromol g(-1) pre-exercise), and not at all in cortisol-treated fish (1.0+/-0.5 micromol g(-1) at 4 h). The rapid glycogen resynthesis in the metyrapone-treated fish was associated with a more rapid inactivation of Phos a and stimulation of GSase I compared to controls. In cortisol-treated fish, Phos a activity persisted throughout 4 h post-exercise; there was also a significant stimulation of GSase I activity. As a consequence of dual activation of Phos a and GSase I, glycogen cycling probably occurred, thus preventing net synthesis. This explains why the post-exercise elevation of cortisol inhibits net glycogen synthesis in trout muscle.

Verapamil reverses abnormal [Ca2+]i and carbohydrate metabolism of PMNL of dialysis patients.

The basal levels of cytosolic calcium ([Ca2+]i) of polymorphonuclear leukocytes (PMNL) are elevated in hemodialysis (HD) patients, and this abnormality has been implicated in the dysfunction of the PMNL of these patients. The elevated [Ca2+]i appears to be due to PTH-induced entry of calcium into PMNL, an action that may be prevented by calcium channel blockers. We examined [Ca2+]i and carbohydrate metabolism of PMNL of normal subjects and of HD patients before, after eight to nine weeks of verapamil therapy (120 mg/day), and after eight to ten weeks of discontinuation of verapamil treatment. In HD patients, the basal levels of [Ca2+]i of PMNL are elevated and their glucose uptake, the activity of total and active forms of glycogen synthetase, and glycogen content are reduced compared to values in normal subjects (P < 0.01). These derangements were normalized after verapamil therapy and re-emerged after discontinuation of treatment with verapamil despite no change in blood levels of PTH. The results indicate that the elevation in [Ca2+]i of PMNL and the consequent derangements in carbohydrate metabolism of these cells are treatable with a calcium channel blocker. The data assign a valuable role for calcium channel blockers for the amelioration of some aspects of cell dysfunction of uremia.

Impairment of glucose tolerance: mechanism of action and impact on the cardiovascular system.

Macrovascular disease, especially coronary heart diseases, have been found to be linked to glucose intolerance. Insulin resistance in respect to glucose uptake in peripheral tissues seems to play an important role in the development of glucose intolerance, since subjects with coronary heart disease mainly are hyperinsulinemic. Insulin resistance may induce not only glucose intolerance but also hypertension, obesity, and dyslipoproteinemia (high very low-density lipoprotein and low high-density lipoprotein values), all variables that add to the risk of coronary heart disease. On the basis of these findings, a new syndrome has been postulated-syndrome X. This syndrome may be caused by inherited insulin resistance in skeletal muscles, and secondary to that arterial hypertension, obesity, and dyslipoproteinemia may develop. Insulin resistance in noninsulin-dependent diabetic persons and in hypertensive subjects is located in skeletal muscles, where insulin's ability to promote nonoxidative glucose metabolism is reduced. The key enzyme in this pathway, glycogen synthase, is proposed as the causal defect responsible for the insulin resistance state, at least in noninsulin-dependent diabetic patients. The pill (sex steroids) may induce a clinical situation that is similar to syndrome X. However, it is important to emphasize that many more studies are needed to substantiate these hypothetical mechanisms behind coronary heart disease.

Abnormalities of carbohydrate metabolism in spontaneously hypertensive rats.

The present study was performed to investigate as to whether peripheral insulin resistance exists in spontaneously hypertensive rats (SHR). After a 12 h fasting period, SHR had significantly higher serum glucose and higher plasma glucagon values in comparison to normotensive control rats (WKY). There was a tendency for higher serum insulin concentrations as well, but this difference did not reach significance. After oral glucose loading or glucose/insulin administration, serum glucose and insulin levels were also higher in SHR compared to WKY rats. Muscle glycogen and glucose concentrations were identical in fasted SHR and WKY rats. With an oral glucose load or glucose/insulin treatment there was a significant increase in muscle glycogen, whereas glucose values declined in skeletal muscle. Both total (a+b-form) phosphorylase activity as well as the active a-form of the enzyme were similar in skeletal muscle of SHR and WKY rats. Glucose/insulin administration or oral glucose loading induced a considerable reduction of both a+b-form and a-form activities. The decrease in muscle phosphorylase activities was almost identical in both groups of animals. There was also a comparable activity of muscle glycogen synthetase activity in all groups of rats. Despite subtile changes of glucose, glucagon and to a lesser degree insulin levels which would be suggestive of insulin resistance, the data obtained from skeletal muscle argue against peripheral insulin resistance in spontaneously hypertensive rats.

Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes.

Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase.

Effect of tourniquet ischaemia on carbohydrate metabolism of dog skeletal muscle.

Metabolic changes in blood and skeletal muscle of dogs before, during and after tourniquet ischaemia were investigated to obtain further information on cellular metabolic abnormalities and restitution during and following long-lasting blood flow interruptions. Total carbohydrate and glycogen contents in the muscle tissue fell during ischaemia and remained significantly decreased even 1 h after recirculation due to inhibition of glycogen synthetase activity. Muscle glucose concentration remained stable during ischaemia and was significantly elevated 1 h after tourniquet release. In contrast, muscle lactate concentration was elevated during ischaemia and normal after recirculation. Blood lactate, pyruvate and serum inorganic phosphate concentrations increased markedly after tourniquet release and were still significantly elevated 1 h after recirculation, whereas ketone bodies and citric acid cycle intermediates remained unchanged. Tourniquet ischaemia had no effect on muscle phosphate concentration or on the activities of proteases, protease inhibitors or hydrolases in the blood. Nevertheless, our results clearly indicate metabolic abnormalities in the blood and skeletal muscle during 5 h of tourniquet ischaemia and even after 1 h of recirculation.

The polymorphonuclear leukocyte in diabetes mellitus.

In polymorphonuclear leukocytes from severely diabetic patients the rate of glycolysis is decreased due to decreased activity of phosphofructokinase, and the glycogen content and rate of glycogen synthesis are decreased due to a decreased total activity of glycogen synthase and an impaired activation of this enzyme. Covalent modification of glycogen synthase by phosphorylation creates a continuum of phosphorylated enzyme forms of decreasing activity. Phosphorylation of a single peptide, whether by the synthase kinase or the cyclic AMP dependent protein kinase, is critical for the associated kinetic changes during the initial phosphorylation. Conversely, dephosphorylation of this particular peptide is associated with complete activation. The protein phosphatase activity of the microsomal fraction may be separated into functionally and possibly also structurally different phosphorylase- and synthase-phosphatase activities, where the latter appears to be dependent on free cytoplasmic Ca2+. It is hypothesized that it is synthase-phosphatase activity that is absent in leukocytes from diabetic patients and is restored upon insulin treatment.

Dephosphorylation of glycogen synthase from human polymorphonuclear leukocytes.

Leukocyte glycogen synthase (UDPglucose:glycogen 4-alpha-d-glucosyltransferase, EC 2.4.1.11) was phosphorylated to about one P1/synthase subunit by either the cAMP-dependent protein kinase or the cAMP-dependent synthase kinase. The relationship between dephosphorylation and the increase in the ratio of independence was investigated by analysis of the release of 32P-labelled phosphopeptides from the trypsin-sensitive and the trypsin-insensitive regions. The trypsin-insensitive region was predominantly dephosphorylated and a close correlation between dephosphorylation of a phosphopeptide in the trypsin-insensitive region and activation of glycogen synthase is reported for the enzyme phosphorylated in both ways.

Activation of the glycogenolytic cascade in human polymorphonuclear leucocytes by different phagocytic stimuli.

Human polymorphonuclear leucocytes were found to respond to activation by immunoglobulin opsonized latex particles and to complement opsonized zymosan particles with a rapid transient increase in cAMP concentration, dissociation of the cAMP dependent protein kinase, activation of glycogen phosphorylase and glycogen break down. However, since phosphorylase kinase was not activated, the activation of phosphorylase is believed to be secondary to non-covalent activation of phosphorylase kinase by Ca2+. Activation by the soluble stimulator phorbol myristate acetate resulted in activation of phosphorylase and glycogen break down, whereas no changes in cAMP concentration, protein kinase activity, or phosphorylase kinase activity were observed. The activation of phosphorylase is ascribed to an increase in cytosolic Ca2+ concentration. The response to stimulation by zymosan was strongly inhibited by ethylene glycol-bis-(beta-aminoethyl ether)-N,N1-tetraacetic acid, which did not affect stimulation by either latex particles or phorbol myristate acetate. The same differential effect of ethylene glycol-bis(beta-aminoethyl ether)-N,N1-tetraacetic acid was observed when the response of the cells was measured as increase in oxygen consumption and activation of the hexose monophosphate shunt.

Effect of glycogenolytic agents on glycogen synthase activity in polymorphonuclear leukocytes. Evidence for a Ca2+-mediated regulation of glycogen synthase activity.

Human polymorphonuclear leukocytes were found to respond to the beta-receptor activators, adrenalin and isoproterenol, with a rapid transient increase in cyclic AMP, activation of cyclic AMP-dependent protein kinase, phosphorylase kinase, deactivation of glycogen synthase and glycogen breakdown. This response was unaffected by the presence of 10 mM EGTA. Incubation of leukocytes with phorbol myristate acetate, which stimulates the hexose monophosphate shunt by a Ca2+ mediated mechanism, resulted in activation of phosphorylase without affecting cyclic AMP-dependent protein kinase or phosphorylase kinase activity, thus indicating a Ca2+-mediated activation of phosphorylase. This was, however, unaffected by EGTA. Prolonged incubation with phorbol myristate acetate was found to result in a parallel activation of phosphorylase and glycogen synthase secondary to a pronounced depletion of cellular glycogen. Addition of glucose to polymorphonuclear leukocytes resulted in total conversion of phosphorylase a to the b form and activation of glycogen synthase, however, when EGTA was included, the response to glucose was greatly amplified, thus indicating the synthase conversion is regulated by Ca2+ sensitive mechanisms which do not involve phosphorylase kinase. Addition of adrenalin to cells previously activated by glucose resulted in an increase in the concentration of cyclic AMP and activation of cyclic AMP-dependent protein kinase but deactivation of synthase was not effectuated under these conditions.

Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes.

Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent alpha-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (Kc equals 0.2-0.3 mM Glc-6-P) were obtained when 1 Pi/subunit was introduced by cAMP dependent protein kinase, 0.5 Pi/subunit by synthase kinase and 0.8 Pi/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites. In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in Kc for Glc. 6-P to 4-5 mM at 1.85 Pi/subunit and to 20 mM at 3.3 Pi/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25). The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 Pi/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 Pi/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant. The rate of decrease in RI in 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and Pi. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.

The hysteretic properties of glycogen synthase I.

Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent process (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 degrees C, the half-time of activation t 1/2 decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The Km for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process. The activation can be described by vt = vf + (vo - vf) e-kt where vt, vf and vo are velocities at times t, O and infinity and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple biolecular process: enzyme + glycogen in equilibrium enzyme-glycogen complex, with the dissociation constant Ks = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.

Abnormal platelet glycogen metabolism in multiple myeloma patients.

This study presents evidence on the occurrence of an inverse relationship between activation of glycogen synthetase and inactivation of phosphorylase in a platelet preparation in vitro. The activities of glycogen synthetase and phosphorylase and the pattern of changes in these activities in platelets from controls and multiple myeloma patients were compared. Platelets obtained from multiple myeloma patients were shown to have an increased glycogen content, accompanied by an elevated level of glycogen synthetase a and a decreased activity of phosphorylase a. The pattern of changes in these enzyme activities during incubation was also different in platelets of controls and multiple myeloma patients. Extracts from patients' platelets prevented glycogen synthetase activation and phosphorylase inactivation of control platelets. Preincubation of platelets from multiple myeloma patients in control plasma resulted in an increased rate of glycogen synthetase activation, and abolished activation of phosphorylase which was found after preincubation in autologous plasma.