Liver Glycogen Phosphorylase Activity Research Articles

Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog Rana sylvatica

We have examined the effect of glucose and caffeine inhibition on the activity of liver glycogen phosphorylase a from the freeze-tolerant frog Rana sylvatica. Kinetic studies indicate that this enzyme exhibits similar sensitivity to glucose inhibition (glucose dissociation constant = 12.5 mM) as the mammalian enzyme. Little inhibition (less than 25%) was observed at normal glucose concentrations (1-5 mM), while significant inhibition (60-95%) occurred at glucose concentrations (50-500 mM) present in freezing-exposed animals. These results favour the hypothesis that in the normal state glucose regulates phosphorylase activity primarily through the promotion of dephosphorylation of phosphorylase a, whereas during freezing regulation is achieved through phosphorylase a inactivation. The caffeine dissociation constant (0.93 mM) and the degree of synergism between caffeine and glucose (interaction factor, alpha = 0.14) were also similar to that observed for the mammalian enzyme. Hence, if a caffeine-like ligand exists in vivo, it must be in low enough amounts during freezing to allow sufficient phosphorylase a activity for high glucose production.

Preliminary studies on carbohydrate metabolism changes in domesticated rainbow trout ( Oncorhynchus mykiss) transferred to diluted seawater (12 p.p.t.)

1. 1. Assessment of glycogen and glucose content and glycogen phosphorylase, glycogen synthetase, hexokinase and fructose 1,6-bisphosphatase activity in liver and white muscle of domesticated rainbow trout ( Oncorhynchus mykiss) transferred to diluted seawater (12 p.p.t.). Plasma glucose levels were also determined. 2. 2. A decline in liver glycogen content was observed throughout the 12–120 hr period in diluted seawater (dSW). Liver free glucose also showed a decrease, though only significant at 120 hr in dSW. 3. 3. The activity of liver glycogen phosphorylase a and a + b was higher in dSW animals, but glycogen synthetase was unchanged. 4. 4. No changes in liver hexokinase and fructose 1,6-biphosphatase activity were observed. 5. 5. In white muscle and plasma only white muscle glycogen synthetase activity showed a change, in particular a decline in dSW animals. 6. 6. Although domesticated rainbow trout do not naturally migrate to seawater, these data suggest that a depletion of liver reserves occurred in a way similar to that described for other salmonids.

Changes in the activity of enzymes, participating in glycogen metabolism of alloxan diabetic rats

Alloxan diabetes induced in white rats by intraperitoneal injection of alloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished. AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities. The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.

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

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 less than 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.

Exercise Training and Dietary Chromium Effects on Glycogen, Glycogen Synthase, Phosphorylase and Total Protein in Rats

The effects of exercise training and dietary chromium intake on rat liver and muscle glycogen metabolism, tissue and body weight and feed consumption were examined. After 16 wk of training, liver, gastrocnemius and biceps femoris glycogen concentrations were higher in the trained compared to sedentary groups, independent of dietary chromium. There was a chromium x training interaction on glycogen synthase activities in the liver and gastrocnemius muscle. Liver glycogen phosphorylase activities (expressed per g liver) were lower in the chromium-supplemented rats as compared to the non-supplemented rats after 5 wk of dietary treatment, but were similar after 8 wk and higher after 18 wk. Gastrocnemius phosphorylase activity (expressed per mg protein) was lower in the trained rats as compared to the sedentary rats after 16 wk, independent of dietary chromium. Biceps femoris phosphorylase activities were not altered due to training or dietary chromium. Total protein concentration increased in the liver but decreased in the gastrocnemius due to dietary chromium. In summary, liver glycogen synthase and phosphorylase activities were dependent upon dietary chromium. Dietary chromium altered gastrocnemius synthase, but not phosphorylase activities. Changes in enzyme activities may be related to the chromium-dependent effects on liver protein and the chromium and training-dependent effects on gastrocnemius total protein.

Regulation of liver metabolism by enzyme phosphorylation during mammalian hibernation.

Kinetic properties of regulatory enzymes of glycolysis in liver of the mouse, Zapus hudsonius, were modified during hibernation, the probable mechanism being covalent modification. Liver glycogen phosphorylase activity was strongly depressed during both short (less than 24 h) and long (5-8 days) term hibernation, the mechanism involving a decrease in both the percentage of enzyme in the active a form and the total amount (a + b) of enzyme expressed. Phosphofructokinase showed kinetic changes (a 2.5-fold increase in Ka for fructose-2,6-P2, 4- and 3.7-fold decreases in I50 values for ATP and citrate, compared to euthermic controls) in liver of hibernators indicative of phosphorylation inactivation of the enzyme. Measured levels of fructose-2,6-P2 in liver did not change during hibernation. Changes in pyruvate kinase kinetics in liver from long term hibernators similarly indicated enzyme phosphorylation in the depressed state (Ka for fructose-1,6-P2 increased 4.4-fold, I50 for L-alanine decreased 6.3-fold). Apparent covalent modification of glycolytic enzymes during hibernation may serve two functions: depression of glycolytic activity as part of the general metabolic rate depression of hibernation, or reorganization of fuel use in the hibernating state to limit carbohydrate catabolism and promote gluconeogenesis.

An early effect of estradiol at hepatic level, previous to its protein synthesis activation

1. 1. Liver glycogen phosphorylase activity is increased before protein syntheis activation by estradiol. 2. 2. This effect is not blocked by antibiotics (actinomycin D and cycloheximide) inhibitor of protein synthesis. 3. 3. At times very similar to those of phosphorylase activation, cAMP levels are not enhanced, as would be expected, but slightly depleted. 4. 4. At similar times, cGMP levels are dramatically increased.

Route of administration of pentobarbital affects activity of liver glycogen phosphorylase.

Liver phosphorylase a activity in intact animals is mostly determined during anesthesia. The aim of this study was to investigate the effect of administering pentobarbital by different routes on activity of liver phosphorylase a. Rats had chronically implanted venous catheters and received pentobarbital (5 mg/100 g body wt) either intraperitoneally, as a slow intravenous infusion, or as an intravenous or intracardial bolus. Times from administration of barbiturate to sampling of the liver were 10 min, 10 min, 85 +/- 32 s (mean +/- SE), and 53 +/- 10 s, respectively. Phosphorylase a activity in % of total phosphorylase activity was 40 +/- 2, 56 +/- 4, 82 +/- 3, and 92 +/- 2, respectively, all significantly different. Thus the route of administration of pentobarbital affects the phosphorylase a activity and should be considered when evaluating this activity. This fact can only be partially explained by differences in duration before the drug takes effect. It is proposed that intraperitoneal injection of pentobarbital may anesthetize hepatic sympathetic nerves or have a direct inhibiting effect on phosphorylase a activity.

Freeze tolerant frogs: cryoprotectants and tissue metabolism during freeze–thaw cycles

Tissue-specific metabolism in the freeze tolerant frog, Rana sylvatica, was monitored over a course of 3 days of freezing exposure at −2.5 °C followed by 11 days of thawing at 3 °C. Freezing induced the rapid accumulation of glucose as a cryoprotectant with maximal levels of 257 μmol/mL in blood and 188, 140, 74, and 28 μmol/g wet weight in liver, heart, kidney, and leg muscle, respectively. Liver showed the fastest rate (20 μmol g−1 h−1 over the initial 7 h) and the shortest half time (5 h) for glucose accumulation. This plus the activation of liver glycogen phosphorylase and a 593 μmol/g decrease in liver glycogen indicated this organ as the major site of cryoprotectant production during freezing. Glucose accumulation was reversed when frogs were thawed (half time for clearance varying from 1.9 days in liver to 8.1 days in leg muscle) with glucose restored as liver glycogen. Extracellular freezing was also accompanied by a progressive accumulation of lactate and alanine as glycolytic end products indicating a reliance on fermentative metabolism during freezing and by a decline in energy status (ATP content and energy charge decrease) in tissues. Relative amounts of lactate and alanine accumulated varied dramatically between tissues: from heart, which produced only lactate, to leg muscle, which had an alanine:lactate ratio of 4.5:1. No other amino acids accumulated in tissues during freezing exposure, but alanine accumulation in leg muscle was inversely proportional to a decline in tissue aspartate, glutamate, and glutamine contents. When thawed, lactate and alanine were readily cleared from tissues and energy status was slowly restored.

Aberrations in the diurnal rhythms of plasma glucose, plasma insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities in genetically diabetic (db/db) mice.

The diurnal rhythms of plasma glucose, insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities were determined in control and genetically diabetic (db/db) mice 8 weeks of age. The diabetic mice showed wide fluctuations in their plasma glucose levels, although being similar to controls near the end of the light period. Little variation was observed in their elevated plasma insulin levels. Liver glycogen levels in diabetic mice were not depleted to the low levels seen in controls during the last part of the light period but were maintained at significantly higher levels. However, maximum attained glycogen levels were similar in the two groups of mice. Alterations were also observed for the diurnal rhythms of glycogen synthase and phosphorylase activities, although again the daily maximums were similar in control and diabetic mice. These findings suggest that the reported changes of several of these metabolic parameters in the db/db mouse may be due to alterations in the diurnal pattern rather than to absolute changes.

Epinephrine effect on carbohydrate metabolism in isolated and perfused catfish liver

Two doses of epinephrine were infused for 6 hr into isolated catfish liver previously perfused with a glucose-free medium or with a medium containing 10 m M glucose. The hormone induced (a) a continuous decrease in liver glycogen level, both in absence and in presence of glucose in the medium; low dose of epinephrine was without effect on the decay of glycogen; (b) a great release of glucose, both in absence and in presence of glucose in the medium; the low dose of epinephrine induced an effect similar to the maximal dose, only in experiments without glucose in the medium; (c) no effect on lactate uptake by liver; or (d) a prevention of the decline in liver glycogen phosphorylase activity observed during 1 hr incubation of liver slices. It has been concluded that epinephrine caused an increase of glucose in perfusion medium with different mechanisms according to the level of glucose and the dose of epinephrine. High doses of hormone cause the glycogenolysis by activation of glycogen phosphorylase, both in presence and in absence of glucose; low doses of epinephrine probably preferentially promote in liver the gluconeogenetic process in glucose-free experiments.

An interaction of testosterone with cell membranes.

The existence of an early effect of testosterone, prior to the effects dependent upon interaction between the hormone-citosolic receptor complex and the cellular nucleus, has been explored in the present paper, in 3-day old chickens. Liver glycogen phosphorylase activity is increased before protein synthesis activation, and furthermore this effect is not blocked by antibiotics (actinomicin D and cycloheximide) inhibitor of protein synthesis. When liver phosphorylase is activated, cAMP levels are not enhanced, as would be expected, but deeply depleted. The hypothesis of a phosphorylase-kinase activation due to an increase in the intracellular Ca++ concentration is considered.

Glycogenolysis in liver of phosphorylase kinase-deficient rats during liver perfusion and ischaemia

Liver glycogen degradation and phosphorylase activity were measured in normal and phosphorylase kinase-deficient (gsd/gsd) rats. During perfusion or ischaemia, gsd/gsd-rat livers showed a brisk glycogenolysis. There was also a small (1.9-fold) but significant transient increase in their phosphorylase alpha activity during ischaemia, despite their phosphorylase b kinase deficiency; it seems unlikely, however, that this was the main determinant of the glycogenolysis.

Fasting reduces the response of liver glycogen phosphorylase to physiological levels of epinephrine in rats

The effect of 24 h fasting on the response of rat liver glycogen phosphorylase activity to an i.v. bolus of 2.75, 5.50 or 22.00 nmol kg −1 of epinephrine was studied. Even the lowest dose increased activity of the a form of the enzyme in the liver of anesthetized, fed rats to approximately 70–80 % of total enzyme activity two minutes after administration. Further increased epinephrine doses failed to potentiate the enzyme response significantly, but shortened the time necessary for attaining the response, and delayed the return of enzyme activity to control values. No activation of phosphorylase was demonstrable after 2.75 nmol kg −1 of the hormone injected to fasted rats, but after increasing the hormone dose to 5.50 nmol kg −1 the enzyme response was the same as in the corresponding fed group at 2 min, and after administering the highest dose both at 1 and 2 min. According to these results, an increased threshold to epinephrine should be added to the already described effects of fasting, i.e. decreased phosphorylase a and total enzyme activity and shortened response to catecholamines. The efficacy of the i.v. bolus of 5.50 nmol kg −1 of epinephrine in increasing plasma epinephrine level to the theoretical value of 27.5 pmol ml −1 was proven by measuring plasma epinephrine which increased during the first minute after hormone administration to 24.5 + 5.9 pmol ml −1, to decrease during an additional minute with a half life of cca 22.2 seconds.

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states.

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.

The Effects of Growth Hormone, Thyroxine and Insulin on the Activities of Reduced Nicotinamide Adenine Dinucleotide Phosphate Dehydrogenase, Glucose-6-Phosphatase and Glycogen Phosphorylase in Fetal Rat Liver

Growth hormone (GH), thyroxine (T4) and insulin were injected, in utero into 20.5 day-old rat fetuses to study the effects of these hormones on the activities of liver NADPH dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase. It was found that at 21.5 days of gestation, GH increases the fetal liver glucose-6-phosphatase activity and decreases the liver glycogen phosphorylase activity. T4 treatment augments the activity of NADPH dehydrogenase even at 0.3% of the dose shown previously to produce premature elevation of activity. Prior to this experiment T4 in large doses has been shown to be capable of elevating glucose-6-phosphatase. However, at the lower T4 dose used, no treatment effect was observed. The fetal rat liver is responsive to insulin at 21.5 days and insulin was able to depress glucose-6-phosphatase activity. Thereby, showing that the influence of insulin on this enzyme begins prior to birth instead of just subsequent to birth.

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rate is blocked by protein synthesis inhibitor

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3′,5′-monosphosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity. In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.

Adrenoceptor-mediated activation of liver glycogen phosphorylase: effects of thyroid state

The effects of altered thyroid state on the adrenergic activation of rat liver glycogen phosphorylase were studied in hepatocytes isolated from normal, thyroidectomized and thyroxine-treated rats. Basal phosphorylase a activity was similar in the three groups. In normal rats, the order of potency of agonists was adrenaline > phenylephrine > isoproterenol, and the effect of adrenaline was blocked by dihydroergocryptine (10 −5M) or phenoxybenzamine (10 −5M) but not by propranolol (10 −5M). These results indicate that in the normal rat activation of this enzyme occurs via α-adrenoceptors. Thyroxine treatment increased sensitivity to all three agonists but did not change their relative potencies. Thyroidectomy dramatically altered the pattern of activation by agonists so that the order of potency now was isoproterenol > adrenaline > phenylephrine and activation by adrenaline was inhibited only by propranolol and not by α-antagonists. Activation of phosphorylase was now mediated via β-adrenoceptors. The effect of thyroidectomy on the adrenoceptor response pattern of the liver develops slowly (more than 2 weeks) and is opposite to the shift from β to α previously observed in the heart. (Br. J. Pharmacol. 59:177, 1977). These findings indicate that hypothyroidism in rats changes activation of liver glycogen phosphorylase from α to a β- type response and that the direction of thyroid-dependent changes in receptor balance is tissue-dependent.

Increases in rat liver cyclic AMP and glycogen phosphorylase activity caused by the herbicide atrazine

The in vivo effects of the triazine herbicide atrazine on rat liver glycogen metabolism was investigated. After administration of atrazine, liver c-AMP was elevated up to threefold, the activity of glycogen phosphorylase increased, the content of glycogen in the liver decreased and the level of blood glucose increased. Cyanuric acid, although not a herbicide but a simple triazine, did alter neither liver c-AMP nor phosphorylase activity. In vitro adenylate cyclase was not activated or inhibited by triazine. However, phosphodiesterase was inhibited non-competitively by the herbicide (0.1–0.4 mM), resembling theophylline in this respect. When atrazine was added to isolated hepatocytes the effect of glucagon-stimulated c-AMP accumulation was potentiated. Thus, atrazine can inhibit phophodiesterase in vitro and in the intact cell. This phophodiesterase inhibition may also occur in vivo and this may lead to the observed effects on glycogen metabolism. However, additional hormonal and/or nervous alterations caused in vivo by the herbicide can not be excluded. No changes in the activities of adenylate cyclase and of phosphodiesterase could be detected in response to in vivo administration of atrazine. However, these negative results may be the consequence of dissociation of the enzyme-atrazine complex during preparation of the liver fractions.