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.     

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.     

Effect of epinephrine infusions imitating plasma epinephrine levels in immobilization stress and of fasting on the activity of key enzymes of liver glycogen metabolism

In adult male rats anaesthetized with pentobarbital, intravenous infusion of 0.25 microgram kg-1 min-1 of epinephrine increased plasma epinephrine levels to more than 10 pmol ml-1 within 5 min, the values remaining constant during the next 20 min. On the other hand, a maximal increase of liver phosphorylase a was found after 5 min and this response was attenuated at 10 and 20 minutes in the fed rats. Fasting for 24 h extinguished the greatest part of this response. During infusion, a decrease of liver synthase a activity was observed in the fasted and of total synthase activity in both the fed and fasted animals. In fed rats an i.v. bolus of 0.50 microgram.kg-1 or doubling of the infusion rate for 5 min, both immediately after 20 min of infusion, led to maximal phosphorylase a activity again and to a decrease of synthase a activity. The levels of cAMP in the liver were increased by the infusion in one series and unchanged in another.     

Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice.

In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.     

The role played by hormonal factors in the rapid activation of liver glycogen phosphorylase in traumatized rats

In adult male SPF rats anaesthetized with pentobarbital and subjected to traumatization in revolving Noble-Collip drums for 2 min (= 120 revolutions) maximal increases of liver glycogen phosphorylase activity were observed. In experiments on rats with permanent arterial catheters for blood sampling no posttraumatic increase of plasma norepinephrine and an only slight increase of plasma epinephrine was observed if the animals were traumatized under anaesthesia, in contrast to the considerable increases in the plasma level of both hormones in rats subjected to the injury without anaesthesia. Time and extent of the phosphorylase response of anaesthetized rats after trauma were compared with changes in enzyme activity after i.v. administration of exogenous epinephrine or glucagon. A nearly maximal response after 1 microgram kg-1 epinephrine was present within 1 min, whereas after 0.1 micrograms kg-1 of glucagon there was comparable phosphorylase activation 2 min after administration of the hormone. The plasma renin-angiotensin activity was not increased after injury for 2 min under anaesthesia so that only the increase in plasma vasopressin fitted in with the criteria for possible activators of phosphorylase. An additional role of glucagon also cannot be excluded on the basis of data obtained by the present authors. The increase of phosphorylase activity in this type of stress is ensured by several mechanisms. Moreover, the high effectivity of these hormonal factors in evoking the phosphorylase response even without major activation of the sympathicoadrenal system is underlined.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

Liver glycogen metabolism in endotoxin shock. II. Endotoxin administration increases glycogen phosphorylase activities in dog livers

The effects of E. coli endotoxin administration on hepatic glycogen phosphorylase activities in dogs were investigated. Hepatic glycogen phosphorylase activities in both control and endotoxic dogs were inactivated spontaneously by preincubation of enzyme preparations at 25 degrees C. Total glycogen phosphorylase activity was not significantly altered during preincubation. The activity of glycogen phosphorylase a was increased by 83 and 80% at 1 and 2 hr postendotoxin, respectively, without preincubation; and by 203 and 133% at 1 and 2 hr postendotoxin, respectively, after 30 min preincubation. Without preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 37 to 58% at 1 hr postendotoxin and to 53% at 2 hr postendotoxin. After 30 min preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 10 to 28% at 1 hr postendotoxin and to 20% at 2 hr postendotoxin. The time required for half maximum inactivation of percentage a activity was 16.5, 33, and 24 min for control, 1 and 2 hr postendotoxin, respectively. Although the Vmax and Km for glucose-1-P for total glycogen phosphorylase were not affected by endotoxin administration, the Vmax for glucose-1-P for glycogen phosphorylase a was increased by 57.3 and 42.7% at 1 and 2 hr postendotoxin, respectively, with no change in the Km values. Glucose inhibited glycogen phosphorylase a activity both in control and endotoxin-injected dogs, but the I50 value was increased by 35% in endotoxin-injected (2 hr) dogs. AMP activated glycogen phosphorylase b activity both in control and endotoxin-injected dogs with no change in A0.5 values between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.     

Hormonal regulation of hepatic glycogen synthase phosphatase

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.     

Temporal relationships between isometric force, phosphorylase, and protein kinase activities in vascular smooth muscle

Vascular smooth muscle contractility is tightly coupled to ATP production by intermediary metabolism. To elucidate mechanisms underlying coordination of metabolism and contractility we studied the time course of isometric force, and the activation of phosphorylase and cAMP-dependent protein kinases during stimulation of bovine coronary arterial strips with KCl. Isometric force reached a maximum after 10 min of exposure to 30 mM KCl (ED90) and was sustained throughout the subsequent 20-min period of contraction. In contrast, activation of phosphorylase was biphasic: enzymic activity reached a maximum (176 +/- 10% of control) after 3 min of contraction and then, though remaining above control, activity declined to a lower level (135 +/- 7% of control). However, no change occurred in the activity ratios for cAMP-dependent protein kinase assessed in either the presence (type II isozyme) or absence (type I isozyme) of 0.5 M NaCl. These data suggest that the activation of phosphorylase during K+-induced contraction is independent of the cAMP system. The biphasic activation of phosphorylase may reflect transient changes in the intracellular concentration of Ca2+ or the activation of a phosphatase(s) during the response.     

In vivo glucose-, glucagon-, and cAMP-induced changes in liver glycogen synthase phosphatase activity.

In normal fed rats, glycogen synthase D phosphatase activity in a glycogen pellet preparation was only partially inhibited (approximately 50%) by high concentrations of EDTA. However, the proportion of phosphatase activity inhibited by EDTA was markedly and rapidly (15 s) increased following glucagon or cAMP administration. Epinephrine administration did not alter the proportion of activity inhibited by EDTA. Glucose administration rapidly (2 min) reduced the proportion of synthase phosphatase activity inhibitable by EDTA. That is, the effect of glucose was just the opposite of that produced by glucagon or cAMP. Insulin administration had no effect on phosphatase activity. Synthase phosphatase activity assayed in the absence of EDTA was similar in all groups except for a moderate increase after glucose administration. Addition of Mg2+ completely reversed EDTA inhibition. Phosphorylase phosphatase activity in each group was not modified by addition of EDTA, although the percentage of phosphorylase in the alpha form was higher in glucagon-treated and lower in the glucose-treated animals as expected. These data suggest the presence of rapidly interconvertible forms of either synthase phosphatase or its substrate synthase D, detectable as a change in EDTA inhibitability and subject to glucose and glucagon control.     

Insulin activates 6-phosphofructo-2-kinase and pyruvate kinase in the liver. Indirect evidence for an action via a phosphatase

The effect of insulin on hepatic glucose production has been studied in anesthetized rats in the postabsorptive state. Insulin decreases significantly hepatic glucose production within 5-10 min. It also increases the level of fructose 2,6-bisphosphate, via an increase in the Vmax of 6-phosphofructo-2-kinase and concomitantly decreased the activity of fructose-2,6-bisphosphatase, resulting in a 5-fold increase in the ratio of kinase/phosphatase. Insulin also increased the apparent Kd of pyruvate kinase for phosphoenolpyruvate. The changes in the activity of 6-phosphofructo-2-kinase and pyruvate kinase were measured after separation from possible modulators, and suggest a decrease in their phosphorylation state which cannot be attributed to a decrease in the level of cAMP or in the activity of cAMP-dependent protein kinase since these two parameters were not modified by insulin. In addition, neither the activity of phosphorylase a nor that of glycogen synthase were modified. The data strongly suggest that the increase in the glycolytic rate plays a role in the effect of insulin on hepatic glucose production and that insulin mediates its effect on the activity of these enzymes via one or more phosphatases.     

Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.     

Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.     

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.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.

The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.     

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.     

Effects of myricetin on glycemia and glycogen metabolism in diabetic rats

In our previous study, we found that myricetin, a naturally occurring bioflavonoid, was able to stimulate glucose transport in rat adipocytes and enhance insulin-stimulated lipogenesis. We report here that after 2 days of treatment with myricetin (3 mg/12 h), hyperglycemia in diabetic rats was reduced by 50% and the hypertriglyceridemia that is often associated with diabetes was normalised. Treatment with myricetin increased hepatic glycogen and glucose-6-phosphate content. It increased hepatic glycogen synthase I activity without having any effect on total glycogen synthase nor phosphorylase a activity. It lowered phosphorylase a activity in the muscle. Thus, the hypoglycemic effect of myricetin is likely to be due to its effect on glycogen metabolism. There was no indication of serious hepatotoxicity with myricetin treatment and therefore, myricetin could be of therapeutic potential in diabetes.