Perturbations in carbohydrate metabolism during cypermethrin toxicity in fish, Tilapia mossambica

Sublethal concentrations (0.04 ppm) of cypermethrin induced significant metabolic changes in brain, liver and gill tissues of fish, T. mossambica. While cypermethrin caused depletion in glycogen and pyruvate levels lactate content was elevated in all the tissues. While phosphorylase 'a' and aldolase activity increased, phosphorylase 'b' activity registered a decrease in the present study. A decrease in lactate dehydrogenase activity with increase in lactate levels suggests reduced mobilization of pyruvate into citric acid cycle. Glucose-6-phosphate dehydrogenase activity was also elevated indicating enhanced oxidation through HMP pathway during cypermethrin toxicity. Inhibition of succinate, malate and isocitrate dehydrogenases and cytochrome c oxidase activity indicates impaired oxidation of carbohydrates through citric acid cycle.     

Intensity and duration of exercise effects on skeletal muscle cAMP, phosphorylase, and glycogen

To gain further insights into the mechanisms regulating skeletal muscle glycogenolysis during exercise, glycogen, phosphorylase, and adenosine 3',5'-cyclic monophosphate (cAMP) were determined in fast-twitch white (FTW) and fast-twitch red (FTR) muscle from groups of rats that ran for 0, 5, 10, 15, or 30 min at either 15 or 30 m/min. Glycogen degradation demonstrated an intensity and duration response in both fiber types. cAMP increased in both fiber types by 5 min and remained elevated at all times measured. FTW muscle cAMP levels were independent of both intensity and duration of exercise. FTR muscle cAMP levels were higher from 10 to 30 min at the 30-m/min intensity compared with the 15-m/min intensity. The ratio of the activity of phosphorylase in the presence of 2 mM AMP X 100 (phosphorylase a%) remained elevated at 20-22% independent of intensity and duration in FTW muscle; however, phosphorylase a% demonstrated an intensity and duration effect in FTR muscle. Glycogenolytic rates decreased with time, even though both cAMP and phosphorylase a% remained elevated in both fiber types. These data suggest that cAMP and phosphorylase a activation can be maintained during exercise in skeletal muscle but indicate a dissociation of these factors from glycogenolysis.     

The role of calcium and cyclic adenosine 3',5'-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes.

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of 45Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound 45Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca2+ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca2+ in the extracellular phase.     

The rate of degradation of liver glycogen phosphorylase is specifically decreased in the C57BL/KsJ — db/db mouse

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.     

Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis

1. Glycogen, glucose, lactate and glycogen phosphorylase concentrations and the activities of glycogen phosphorylase a and acid 1,4-alpha-glucosidase were measured at various times up to 120 min after death in the liver and skeletal muscle of Wistar and gsd/gsd (phosphorylase b kinase deficient) rats and Wistar rats treated with the acid alpha-glucosidase inhibitor acarbose. 2. In all tissues glycogen was degraded rapidly and was accompanied by an increase in tissue glucose and lactate concentrations and a lowering of tissue pH. In the liver of Wistar and acarbose-treated Wistar rats and in the skeletal muscle of all rats glycogen loss proceeded initially very rapidly before slowing. In the gsd/gsd rat liver glycogenolysis proceeded at a linear rate throughout the incubation period. Over 120 min 60, 20 and 50% of the hepatic glycogen store was degraded in the livers of Wistar, gsd/gsd and acarbose-treated Wistar rats, respectively. All 3 types of rat degraded skeletal muscle glycogen at the same rate and to the same extent (82% degraded over 2 hr). 3. In Wistar rat liver and skeletal muscle glycogen phosphorylase was activated soon after death and the activity of phosphorylase a remained well above the zero-time level at all later time points, even when the rate of glycogenolysis had slowed significantly. Liver and skeletal muscle acid alpha-glucosidase activities were unchanged after death. 4. The decreased rate and extent of hepatic glycogenolysis in both the gsd/gsd and acarbose-treated rats suggests that this process is a combination of phosphorolysis and hydrolysis. 5. Glycogen was purified from Wistar liver and skeletal muscle at various times post mortem and its structure investigated. Fine structural analysis revealed progressive shortening of the outer chains of the glycogen from both tissues, indicative of random, lysosomal hydrolysis. Analysis of molecular weight distributions showed inhomogeneity in the glycogen loss; in both tissues high molecular weight glycogen was preferentially degraded. This material is concentrated in lysosomes of both skeletal muscle and liver. These results are consistent with a role for lysosomal hydrolysis in glycogen degradation.     

Intensity and duration effects of exercise on heart cAMP, phosphorylase, and glycogen

Glycogen, phosphorylase, and adenosine 3',5'-cyclic monophosphate (cAMP) were determined in rat heart following an acute exercise bout. Intensity and duration of exercise were varied to gain further insights into the mechanism regulating myocardial glycogenolysis during exercise. Groups of rats were run at either 15 or 30 m/min for 0, 5, 10, 15, or 30 min and immediately killed. Heart glycogen degradation was influenced by intensity and duration of exercise and was independent of cAMP levels and activation of phosphorylase to its a form. cAMP levels were increased in the heart, dependent on intensity and duration of exercise. Phosphorylase in the a form increased at the onset of exercise, independent of intensity, and remained elevated throughout the exercise despite little or no glycogenolysis. Absolute phosphorylase a activity was also increased with exercise and was independent of intensity of exercise. Compared with resting levels, total phosphorylase activity was decreased at all times at the lower exercise intensity, whereas total phosphorylase activity declined at the higher intensity only after glycogenolysis had occurred. These data suggest that myocardial glycogen degradation during exercise can occur independently of cAMP and that the percentage of phosphorylase in the a form is not a good indicator of glycogenolytic rate.     

Modulation of Glycogen Phosphorylase Activity Affects 5‐Phosphoribosyl‐1‐Pyrophosphate Availability in Rat Hepatocyte Cultures

The effect of modulation of the rate of glycogenolysis on the availability of 5‐phosphoribosyl‐1‐ pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. Dibutyryl cyclic AMP (dbcAMP), forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%–56%. Arg‐vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, did not affect PRPP availability. dbcAMP, but not phenylephrine, increased the degradation of pre labeled glycogen by 57%. Caffeine and CP‐91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.     

Modulation of glycogen phosphorylase activity affects 5-phosphoribosyl-1-pyrophosphate availability in rat hepatocyte cultures

The effect of modulation of the rate of glycogenolysis on the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. Dibutyryl cyclic AMP (dbcAMP), forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%-56%. Arg-vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, did not affect PRPP availability. dbcAMP, but not phenylephrine, increased the degradation of pre labeled glycogen by 57%. Caffeine and CP-91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.     

The role of calcium and cyclic adenosine 3′,5′-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of ⁴⁵Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound ⁴⁵Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca²⁺ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca²⁺ in the extracellular phase.     

Changes in the rates of glycogenesis, glycogenolysis, lipogenesis, and lipolysis in selected tissues of the coho salmon (Oncorhynchus kisutch) associated with parr-smolt transformation

The rates of glycogenesis, glycogenolysis, lipogenesis and lipolysis were determined in selected tissues of the coho salmon, Oncorhynchus kisutch, during the period of parr-smolt transformation between February 1983 and June 1983. Glycogen synthesis in the liver, measured by uridine diphosphate formation, decreased 54% from initial levels. Liver glycogen phosphorylase a activity increased by 66%. Neutral lipid (sterol) and fatty acid synthesis in the liver and mesenteric fat was measured by tritium incorporation. Fatty acid synthesis in the liver and mesenteric fat decreased by 88% and 81%, respectively, between late February (parrs) and early June (smolts). There was no significant change in the rate of tritium incorporation into liver or mesenteric fat neutral lipids during the sampling period. Lipolytic rates were assessed by measuring the release of 14C-oleic acid from 14C-triolein in the presence of partially purified triacylglycerol lipase enzyme preparations from the liver, dark muscle and mesenteric fat. Liver, dark muscle and mesenteric fat lipase activity increased by 86%, 146% and 289%, respectively, during the sampling period. Increased glycogen and lipid breakdown, and concomitant decreased glycogen and fatty acid synthesis would contribute to the lipid and glycogen depletion observed in salmonid species undergoing parr-smolt transformation.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

Exercise and glycogen depletion: effects on ability to activate muscle phosphorylase

Phosphorylase activation reverses during prolonged contractile activity. Our first experiment was designed to determine whether this loss of ability to activate phosphorylase by stimulation of muscle contraction persists following exercise. Phosphorylase activation by stimulation of muscle contraction was markedly inhibited in rats 25 min after exhausting exercise. To evaluate the role of glycogen depletion, we accelerated glycogen utilization by nicotinic acid administration. A large difference in muscle glycogen depletion during exercise of the same duration did not influence the blunting of phosphorylase activation. Phosphorylase activation by stimulation of contraction was more severely inhibited following prolonged exercise than after a shorter bout of exercise under conditions that resulted in the same degree of glycogen depletion. A large difference in muscle glycogen repletion during 90 min of recovery was not associated with a significant difference in the ability of muscle stimulation to activate phosphorylase, which was still significantly blunted. Phosphorylase activation by epinephrine was also markedly inhibited in muscle 25 min after strenuous exercise but had recovered completely in glycogen-repleted muscle 90 min after exercise. These results provide evidence that an effect of exercise other than glycogen depletion is involved in causing the inhibition of phosphorylase activation; however, they do not rule out the possibility that glycogen depletion also plays a role in this process.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Activation of glycogenolysis in perfused rat livers by glucagon and metabolic inhibitors

The activation of hepatic glycogenolysis by glucagon and metabolic inhibitors was studied in isolated perfused livers from fed rats. Glucose production rates and phosphorylase activity were increased by all these agents. If iodoacetate (1 mM) and cyanide (1 mM) were infused simultaneously, glycogenolysis was activated to the same extent as by glucagon (1 nM). The effects of the hormone were additive to those of cyanide, but not to those of iodoacetate. When glycogen breakdown was maximally activated by cyanide plus glucagon, additional iodoacetate was inhibitory. The glucagon-induced release of cyclic AMP into the perfusate was partially suppressed by iodoacetate. The inhibitors caused various degrees of depletion of the tissue ATP content and parallel augmentation of the AMP levels. ADP rose to a lesser extent. Indirect evidence suggested that of a progressive lowering of the cellular ATP levels was accompanied by an inhibition of enzyme dephosphorylation as well as of phosphorylation processes. However, dephosphorylation appeared to be more sensitive to changes of the energy balance, resulting in an activation of phosphorylase in response to the metabolic inhibitors.     

Activation of hepatic glycogen phosphorylase b in vivo by sodium sulphate in normal (Wistar) and phosphorylase b kinase-deficient (gsd/gsd) rats.

Sulphate ions have been known for some years to enhance the activity of hepatic glycogen phosphorylase b in vitro. Here we report that intravenous injections of 4.92 mmol of Na2SO4/kg body wt. to rats induced marked hepatic glycogenolysis in vivo, accompanied by polyuria, glycosuria and a mild hyperglycaemia. These effects were observed both in normal (Wistar) rats and in gsd/gsd rats that lacked hepatic phosphorylase kinase. In both rat strains the activity of glycogen phosphorylase in liver extracts was enhanced by pretreatment of the animals with Na2SO4, but in phosphorylase kinase-deficient livers the enhancement was solely in phosphorylase b activity, whereas both the a and b forms of the enzyme were activated in normal livers. Hepatic glycogenolysis was also induced by perfusing rat livers, both normal and gsd/gsd, with 25 mM-Na2SO4. Under these conditions both the rat strains showed only enhanced activities of glycogen phosphorylase b. This suggested that the increased activity of phosphorylase a in the extracts of normal livers after Na2SO4 administration in vivo was due to a hormonally mediated conversion of the b form into the a form. The activation of glycogen phosphorylase b was stable to dilution and appeared to be due to a long-lasting structural change in the enzyme or very tight binding of an activator.     

Inability of glucagon to regulate glycogen metabolism in rat hepatocytes isolated after fasting and refeeding high-carbohydrate diets

Studies are described which demonstrate that the ability of glucagon, epinephrine, and dibutyryl-cAMP to stimulate glycogenolysis is impaired in rat hepatocytes isolated from animals starved for 24 h and then refed a sucrose-rich diet or refed standard rat chow. The impaired regulation of glycogenolysis by glucagon was observed within 24 h after refeeding and persisted for at least 3 days. The inability of glucagon to stimulate glycogen breakdown in the refed condition appeared to be due to a suppressed activation of glycogen phosphorylase and phosphorylase b kinase by the hormone. The capacity of glucagon to regulate pyruvate kinase and glycolysis was not altered by refeeding, suggesting that the defect lies beyond interaction of the hormone at its receptor. Prolonged incubation of hepatocytes from refed rats was accompanied by depletion of glycogen reserves and was accompanied by restoration of hormonal stimulation of glycogenolysis. Addition of glycogen to cell-free extracts was found to inhibit phosphorylase b kinase but not phosphorylase. The findings of this investigation are consistent with the interpretation that high levels of glycogen present of liver after refeeding may lead to a diminished activity of phosphorylase b kinase and its hormonal regulation.     

Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts

In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6-phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity.     

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.     

Bioactivity of glycogen phosphorylase inhibitors that bind to the purine nucleoside site

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.     

Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant. A biochemical study.

The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.