Liver Glycogen Phosphorylase Activity Research Articles

A Mouse Model of Glycogen Storage Disease Type IX-Beta: A Role for Phkb in Glycogenolysis.

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, β, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (Phkb−/−) mouse model. In this study, we assessed fasting blood glucose and ketone levels, serum metabolite concentrations, glycogen phosphorylase activity, and gene expression of gluconeogenic genes and fibrotic genes. Phkb−/− mice displayed hepatomegaly with lower fasting blood glucose concentrations. Phkb−/− mice showed partial liver glycogen phosphorylase activity and increased sensitivity to pyruvate, indicative of partial glycogenolytic activity and upregulation of gluconeogenesis. Additionally, gene expression analysis demonstrated increased lipid metabolism in Phkb−/− mice. Gene expression analysis and liver histology in the livers of old Phkb−/− mice (>40 weeks) showed minimal profibrogenic features when analyzed with age-matched wild-type (WT) mice. Collectively, the Phkb−/− mouse recapitulates mild clinical features in patients with GSD-IX-beta. Metabolic and molecular analysis confirmed that Phkb−/− mice were capable of sustaining energy homeostasis during prolonged fasting by using partial glycogenolysis, increased gluconeogenesis, and potentially fatty acid oxidation in the liver.

Tyrosol, a phenolic compound, ameliorates hyperglycemia by regulating key enzymes of carbohydrate metabolism in streptozotocin induced diabetic rats

The present study was designed to evaluate the effects of tyrosol, a phenolic compound, on the activities of key enzymes of carbohydrate metabolism in the control and streptozotocin-induced diabetic rats. Diabetes mellitus was induced in rats by a single intraperitoneal injection of streptozotocin (40mg/kg body weight). Experimental rats were administered tyrosol 1ml intra gastrically at the doses of 5, 10 and 20mg/kg body weight and glibenclamide 1ml at a dose of 600μg/kg body weight once a day for 45days. At the end of the experimental period, diabetic control rats exhibited significant (p<0.05) increase in plasma glucose, glycosylated hemoglobin with significant (p<0.05) decrease in plasma insulin, total hemoglobin and body weight. The activities of key enzymes of carbohydrate metabolism such as phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly (p<0.05) increased and the activities of hexokinase and glucose-6-phosphate dehydrogenase were significantly (p<0.05) decreased in the liver and kidney of diabetic control rats. Further, antioxidants were lowered in diabetic control rats. A significant (p<0.05) decline in glycogen level in the liver and muscle and glycogen synthase activity in the liver and a significant (p<0.05) increase in the activity of liver glycogen phosphorylase were observed in diabetic control rats compared to normal control rats. Oral administration of tyrosol to diabetic rats reversed all the above mentioned biochemical parameters to near normal in a dose dependent manner. Tyrosol at a dose of 20mg/kg body weight showed the highest significant effect than the other two doses. Immunohistochemical staining of pancreas revealed that tyrosol treated diabetic rats showed increased insulin immunoreactive β-cells, which confirmed the biochemical findings. The observed results were compared with glibenclamide, a standard oral hypoglycemic drug. The results of the present study suggest that tyrosol decreases hyperglycemia, by its antioxidant effect.

The Influence of Social Status on Hepatic Glucose Metabolism in Rainbow TroutOncorhynchus mykiss

The effects of chronic social stress on hepatic glycogen metabolism were examined in rainbow trout Oncorhynchus mykiss by comparing hepatocyte glucose production, liver glycogen phosphorylase (GP) activity, and liver β-adrenergic receptors in dominant, subordinate, control, fasted, and cortisol-treated fish. Hepatocyte glucose production in subordinate fish was approximately half that of dominant fish, reflecting hepatocyte glycogen stores in subordinate trout that were just 16% of those in dominant fish. Fasting and/or chronic elevation of cortisol likely contributed to these differences based on similarities among subordinate, fasted, and cortisol-treated fish. However, calculation of the "glycogen gap"--the difference between glycogen stores used and glucose produced--suggested an enhanced gluconeogenic potential in subordinate fish that was not present in fasted or cortisol-treated trout. Subordinate, fasted, and cortisol-treated trout also exhibited similar GP activities (both total activity and that of the active or a form), and these activities were in all cases significantly lower than those in control trout, perhaps reflecting an attempt to protect liver glycogen stores or a modified capacity to activate GP. Dominant trout exhibited the lowest GP activities (20%-24% of the values in control trout). Low GP activities, presumably in conjunction with incoming energy from feeding, allowed dominant fish to achieve the highest liver glycogen concentrations (double the value in control trout). Liver membrane β-adrenoceptor numbers (assessed as the number of (3)H-CGP binding sites) were significantly lower in subordinate than in dominant trout, although this difference did not translate into attenuated adrenergic responsiveness in hepatocyte glucose production in vitro. Transcriptional regulation, likely as a result of fasting, was indicated by significantly lower β(2)-adrenoceptor relative mRNA levels in subordinate and fasted trout. Collectively, the data indicate that social status shapes liver metabolism and in particular glycogen metabolism, favoring accumulation of glycogen reserves from incoming energy in dominant fish and reliance on onboard fuels in subordinate fish.

Effect of glucopyranosylidene-spiro-thiohydantoin on glycogen metabolism in liver tissues of streptozotocin-induced and obese diabetic rats

The major role of liver glycogen is to supply glucose to the circulation in order to maintain normal blood glucose levels. In the muscle and liver, the accumulation and breakdown of glycogen are regulated by the reciprocal activities of glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase catalyses the key step of glycogen degradation and its activity is inhibited by glucose and its analogues. Thus, any readily accessible inhibitor of glycogen phosphorylase may serve as a potential therapy for non-insulin-dependent or type 2 diabetes. Hepatic glycogen phosphorylase has been identified as a novel target for drugs that control blood glucose concentration. Glucopyranosylidene-spiro-thiohydantoin (TH) was found to be one of the most potent glucose derivates, inhibiting the catalytic activity of both muscle and liver glycogen phosphorylase. Here, we demonstrated the co-ordinated regulation of glycogen phosphorylase and synthase by 50 µM TH in liver extracts of Wistar rats, resulting in the activation of synthase by a shortening of the latency compared to control animals. TH was also effective in lowering blood glucose levels and restoring hepatic glycogen content in streptozotocin-induced diabetic rats. Furthermore, intravenous administration of TH to Zucker diabetic fatty rats significantly decreased hepatic glycogen phosphorylase a levels, and the activation of synthase was initiated without any delay.

Hepatic 11 beta-hydroxysteroid dehydrogenase 1 involvement in alterations of glucose metabolism produced by acidotic stress in rat

11 beta-hydroxysteroid dehydrogenase (HSDs) enzymes regulate the activity of glucocorticoids in target organs. HSD1, one of the two existing isoforms, locates mainly in CNS, liver and adipose tissue. HSD1 is involved in the pathogenesis of diseases such as obesity, insulin resistance, arterial hypertension and the Metabolic Syndrome. The stress produced by HCl overload triggers metabolic acidosis and increases liver HSD1 activity associated with increased phosphoenolpyruvate carboxykinase, a regulatory enzyme of gluconeogenesis that is activated by glucocorticoids, with increased glycaemia and glycogen breakdown. The aim of this study was to analyze whether the metabolic modifications triggered by HCl stress are due to increased liver HSD1 activity. Glycyrrhetinic acid, a potent HDS inhibitor, was administered subcutaneously (20 mg/ml) to stressed and unstressed four months old maleSprague Dawley rats to investigate changes in liver HSD1, phosphoenolpyruvate carboxykinase (PECPK) and glycogen phosphorylase activities and plasma glucose levels. It was observed that all these parameters increased in stressed animals, but that treatment with glycyrrhetinic acid significantly reduced their levels. In conclusion, our results demonstrate the involvement of HSD1 in stress induced carbohydrate disturbances and could contribute to the impact of HSD1 inhibitors on carbohydrate metabolism and its relevance in the study of Metabolic Syndrome Disorder and non insulin-dependent diabetes mellitus.

Inhibitory effect of fasting on the activation of liver glycogen phosphorylase of rats by arginine-vasopressin.

The i.v. bolus of 50 ng kg-1 Arginine-vasopressin administered to adult male rats anaesthetized with Pentobarbital (50 mg kg-1 i.p.) increases the activity of liver glycogen phosphorylase in fed and 24 h fasted rats to the same extent; after 25 ng kg-1 of the drug the enzyme response after fasting is barely detectable, whereas in the fed animals a response similar to that after 50 ng kg-1 persists.

FR258900, a Novel Glycogen Phosphorylase Inhibitor Isolated from Fungus No. 138354

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.

Intermittent and recurrent hepatomegaly due to glycogen storage in a patient with type 1 diabetes: Genetic analysis of the liver glycogen phosphorylase gene ( PYGL)

We report a 19-year-old woman who had a history of type 1 diabetes with recurrent glycogen accumulation in the liver. During her infantile period she presented with no hepatomegaly nor growth retardation. On admission she was diagnosed with diabetic ketoacidosis (DKA). She also had hepatomegaly and elevated transaminase levels, but these abnormalities had resolved after administration of insulin. However, 4 weeks after DKA marked hepatomegaly and elevated transaminases were reappeared with simultaneous hypoglycemia which suggested an impaired glycogenolysis in the extraordinary conditions. We supposed the partial deficiency of liver glycogen phosphorylase activity in this patient and analyzed the liver glycogen phosphorylase gene ( PYGL). Deduced amino acid sequence of the PYGL in this patient was completely identical to that reported by Burwinkel et al. (Y15233), however, the nucleotide sequence of PYGL cDNA was heterozygous for substitutions at positions Asp339 ( GA T to GA C ) on exon 9 and Ala703 ( GC T to GC C on exon 17, respectively. These SNPs were also screened in 51 Japanese normal subjects by PCR-based direct sequencing or PCR–RFLP method. The same genotype observed in this patient was detected in 2 of 51(3.9%) normal subjects. These results suggest that the structure of PYGL coding sequence in this patient is unlikely to account for her excessive liver glycogen accumulation. Further studies including genetic analysis on the promoter region of the gene are necessary to clarify the etiology of susceptibility to excessive liver glycogen storage in patients with type 1 diabetes.

Phytosterols affect endocrinology and metabolism of the field vole (Microtus agrestis).

Phytosterols or plant sterols (PS) enter the ecosystem via pulp mill effluents. They are also consumed by the general population of developed countries in natural remedies and margarines to lower elevated serum cholesterol levels. This study screened the endocrine and enzymatic parameters of the field vole (Microtus agrestis) for the effects of subchronic PS exposure at three doses (0, 5, or 50 mg of PS kg(-1) day(-1)). PS at 5 or 50 mg kg(-1) day(-1) decreased the relative liver weight of the voles. The kidney glycogen phosphorylase activity decreased at 5 or 50 mg kg(-1) day(-1), but the liver glycogen phosphorylase activity increased at 5 mg kg(-1) day(-1). The plasma estradiol and testosterone concentrations of males were higher due to PS supplement at 5 mg kg(-1) day(-1). This can be due to increased sex steroid synthesis from PS precursors. Biotransformation enzyme activities were not affected. PS caused multiple, previously unreported effects that were more pronounced at a low dose. As 5 mg PS kg(-1) day(-1) is the recommended dose for various health products, a thorough risk assessment of the effects and interactions of PS is warranted.

Melatonin and the wintering strategy of the tundra vole, Microtus oeconomus.

Short photoperiod induces physiological changes connected to the wintering of the tundra vole, Microtus oeconomus. The aim of the present study was to investigate the effects of continuous melatonin treatment on selected hormones and enzyme activities associated with energy metabolism in the species. Liver, kidney, and muscle glycogen concentrations and glycogen phosphorylase activities, as well as liver and kidney glucose-6-phosphatase and lipase esterase activities were determined. Plasma leptin, ghrelin, thyroxine, testosterone, cortisol, and melatonin concentrations were also measured. Exogenous melatonin stimulated gluconeogenesis, increased glycogen stores, and reduced fat mobilization in kidneys. Melatonin treatment also increased the food intake of the voles. This may have been mediated via elevated ghrelin levels of the melatonin-treated animals, as ghrelin is known to increase appetite of rodents. Winter metabolism of the species does not seem to require accumulation of fat or extra stores of liver or muscle glycogen. On the contrary, successful wintering of the tundra vole presumably depends on continuous food availability.

Effects of acute handling stress on whitefish Coregonus lavaretus after prolonged exposure to biologically treated and untreated bleached kraft mill effluent.

Exposure of fish to water of impaired quality has been shown to disrupt the function of the hypothalamo-pituitary-interrenal (HPI) axis and alter the interpretation of data from field studies due to the varying effects of handling and delayed sampling on exposed and reference animals. In the present study, juvenile whitefish, Coregonus lavaretus, were exposed for 6 weeks to diluted (4-8%) untreated and biologically treated bleached kraft mill effluent (BKME) and their response to acute handling was investigated. Liver microsomal EROD activity and glycogen phosphorylase (GPase) activity, in addition to gill Na+-K+-ATPase activity, and blood hemoglobin and hematocrit levels were increased in whitefish exposed for 6 weeks to untreated BKME, whereas those exposed to treated BKME exhibited increased blood hemoglobin and red blood cell K+ concentrations. Both handling procedures, exposure to a shallow water (10 cm, 5 min) and to an air challenge (10 s air/10 s water/30 s air/10 s water/10 s air), resulted in acute physiological stress, as recorded after 5-, 60-, and 120-min recovery periods. Following air exposure, the levels of plasma cortisol, blood glucose, hemoglobin, and hematocrit as well as the liver GPase activity were increased, and liver glycogen concentration decreased in control fish. These responses were attenuated in fish exposed to untreated or treated BKME. Plasma estradiol and testosterone levels were not affected by the BKME exposures or by the air challenge. Handling also resulted in attenuated EROD induction in fish exposed to untreated BKME. According to the present findings, the sensitivity of some widely used cellular and physiological variables may be improved by time-dependent standardization when interpreting data obtained following delayed sampling.

Activation of Human Liver Glycogen Phosphorylase by Alteration of the Secondary Structure and Packing of the Catalytic Core

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.

Activation of Human Liver Glycogen Phosphorylase by Alteration of the Secondary Structure and Packing of the Catalytic Core

Activation of Human Liver Glycogen Phosphorylase by Alteration of the Secondary Structure and Packing of the Catalytic Core

Liver Hyperplasia and Paradoxical Regulation of Glycogen Metabolism and Glucose-sensitive Gene Expression in GLUT2-null Hepatocytes: FURTHER EVIDENCE FOR THE EXISTENCE OF A MEMBRANE-BASED GLUCOSE RELEASE PATHWAY

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5–12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.

Gram-scale synthesis of a glucopyranosylidene-spiro-thiohydantoin and its effect on hepatic glycogen metabolism studied in vitro and in vivo

A high yielding, simple synthesis is described starting from d-glucose to produce gram quantities of a glucopyranosylidene-spiro-thiohydantoin. This compound efficiently inhibited the activity of rat liver glycogen phosphorylase a; moreover, it also activated phosphorylase phosphatase which, in turn, decreased the amount of glycogen phosphorylase a. Both effects result in the inhibition of glycogen mobilization and the formation of glucose from glycogen.

Effects of dichloroacetate on glycogen metabolism in B6C3F1 mice

Dichloroacetate (DCA) is a by-product of drinking water chlorination. Administration of DCA in drinking water results in accumulation of glycogen in the liver of B6C3F1 mice. To investigate the processes affecting liver glycogen accumulation, male B6C3F1 mice were administered DCA in drinking water at levels varying from 0.1 to 3 g/l for up to 8 weeks. Liver glycogen synthase (GS) and glycogen phosphorylase (GP) activities, liver glycogen content, serum glucose and insulin levels were analyzed. To determine whether effects were primary or attributable to increased glycogen synthesis, some mice were fasted and administered a glucose challenge (20 min before sacrifice). DCA treatments in drinking water caused glycogen accumulation in a dose-dependent manner. The DCA treatment in drinking water suppressed the activity ratio of GS measured in mice sacrificed at 9:00 AM, but not at 3:00 AM. However, net glycogen synthesis after glucose challenge was increased with DCA treatments for 1–2 weeks duration, but the effect was no longer observed at 8 weeks. Degradation of glycogen by fasting decreased progressively as the treatment period was increased, and no longer occurred at 8 weeks. A shift of the liver glycogen–iodine spectrum from DCA-treated mice was observed relative to that of control mice, suggesting a change in the physical form of glycogen. These data suggest that DCA-induced glycogen accumulation at high doses is related to decreases in the degradation rate. When DCA was administered by single intraperitoneal (i.p.) injection to naı̈ve mice at doses of 2–200 mg/kg at the time of glucose challenge, a biphasic response was observed. Doses of 10–25 mg/kg increased both plasma glucose and insulin concentrations. In contrast, very high i.p. doses of DCA (>75 mg/kg) produced progressive decreases in serum glucose and glycogen deposition in the liver. Since the blood levels of DCA produced by these higher i.p. doses were significantly higher than observed with drinking water treatment, we conclude that apparent differences with data of previous investigations is related to substantial differences in systemic dose and/or dose–time relations.

Analogous activation of bovine liver glycogen phosphorylase by AMP and IMP

The mechanism of activation of glycogen phosphorylase is incompletely understood, although adenosine and inosine nucleotides are known to be important allosteric activators. In this study the activation of glycogen phosphorylases a and b from bovine liver by adenosine 5′-monophosphate (AMP) and inosine 5′-monophosphate (IMP) has been investigated and the results compared with the activation of the muscle isozyme by the same nucleotides. Enzyme activity was determined by spectrophotometric measurement of inorganic phosphate produced in the phosphorylase-catalysed reaction of glycogen synthesis. Liver phosphorylase b binds both nucleotides non-co-operatively (Hill coefficients of 1.0 ± 0.1), with changes in the maximum velocity to 75 or 80 μmol min −1 mg −1 in the presence of adenosine 5′-monophosphate or inosine 5′-monophosphate, respectively, but no change in the enzyme affinity towards the substrate, glucose-1-phosphate. Binding of glucose-1-phosphate is co-operative and the kinetic data have been fitted with the Monod-Wyman-Changeux model. Liver phosphorylase a has a maximum velocity similar to that of the b form in the presence of nucleotides. Binding of glucose-1-phosphate to the enzyme is non-co-operative (Hill coefficient of 1.0 ± 0.1) and the affinities in the presence of the nucleotides (Michaelis constants of 28 ± 0.2 mM or 27 ± 0.2 mM for adenosine 5′-monophosphate or inosine 5′-monophosphate) are stronger than those of the b form. It is concluded that the activity of bovine liver phosphorylase a and b is similarly influenced by adenosine 5′-monophosphate and inosine 5′-monophosphate. The b form seems to behave like muscle phosphorylase b in response to inosine 5′-phosphate; however, the binding of adenosine 5′-phosphate does not induce the conformational change necessary to activate the liver enzyme, as occurs with the muscle isozyme.

Insulin regulates liver glycogen synthase and glycogen phosphorylase activity reciprocally in rhesus monkeys.

In skeletal muscle of both humans and monkeys, the effects of in vivo insulin during a euglycemic hyperinsulinemic clamp on the enzymes and substrates of glycogen metabolism have been well established. In liver, such effects of insulin during a clamp have not been previously studied in primates. To examine insulin action at the liver, euglycemic hyperinsulinemic clamps were performed in 10 lean young adult male rhesus monkeys. Liver biopsies were obtained at three time points: basal (fasting), that is, immediately before the onset of the clamp, and during insulin infusion at 130 and 195 min. Glycogen synthase (GS), glycogen phosphorylase (GP), glucose 6-phosphate (G-6-P), and glycogen were determined at each time point, with the greatest effects observed most frequently at 195 min. Whole body insulin-mediated glucose disposal rate was related to the change in the independent activity of GS (r = 0.63, P < 0.05). Insulin increased the GS fractional activity (P < 0.005) and decreased the activity ratio of GP (P < 0.001) compared with basal. The changes in fractional activity of GS and in activity ratio of GP were inversely related (r = - 0.68, P < 0.05), G-6-P concentration was decreased during insulin stimulation compared with basal (P = 0.01). Glycogen concentration was not significantly different between the basal and insulin-stimulated time points. We conclude that insulin during a euglycemic clamp activates liver GS while inhibiting liver GP and that insulin action on liver GS is positively related to whole body insulin-mediated glucose disposal rates in lean young adult rhesus monkeys.

β-Adrenergic, Hormonal, and Nervous Influences on Cryoprotectant Synthesis by Liver of the Freeze-Tolerant Wood FrogRana sylvatica

The effects of various catecholamines, α- and β-adrenergic agonists and antagonists, hormones, hormone receptor blockers, and other inhibitors, as well as destruction of the nervous system by pithing, were analyzed for their ability to modulate the freezing-induced cryoprotectant output by liver of the wood frogRana sylvatica.Of the numerous treatments tried only intraperitoneal injections of glucagon (a glycogenolytic hormone) and propranolol (a blocker of β-adrenergic receptors), both of which modulate hepatic cyclic AMP levels, altered cryoprotectant output. Glucagon significantly enhanced glucose output into the blood during freezing (by 3.5-fold compared with sham-injected frogs), whereas propranolol significantly reduced the hyperglycemia, liver and blood glucose concentrations being reduced by 55 and 42%, respectively, compared with values for untreated frozen frogs. However, treatment with the α-adrenergic blocker phentolamine did not block freezing-induced cryoprotectant output, indicating that Ca2+-mediated mechanisms are not involved in the hyperglycemic response by liver in these frogs. The mechanism of propranolol action was to suppress the activation of liver glycogen phosphorylase so that the amount of active phosphorylaseain liver of propranolol-treated frogs after freezing exposure was only 5% of the corresponding activity in liver of untreated frozen frogs. The β-adrenergic nature of catecholamine regulation of blood glucose levels was confirmed by the ability of the β-adrenergic agonist isoproterenol to mimic the hyperglycemia induced by epinephrine or norepinephrine injection in unfrozen frogs at 3°C, whereas the α-adrenergic agonist phenylephrine was ineffective. The data indicate that the cryoprotectant output response during freezing ofR. sylvaticais mediated by cAMP-dependent mechanisms within the liver under the control of plasma hyperglycemic hormones.