Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.     

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

Fetal mouse tissues on gestational days 14, 16 and 19 were incubated with ¹⁴C-corticosterone (B) and ³H-11-dehydrocorticosterone (A), and the steroids were separated chromatograhically to determine the ratio reduction/dehydrogenation. In placenta the ratio remained high, >6, throughout this period, while it rose from 0.06 to 0.6 in brain and from 0.1 to 0.5 in gut. In liver, the ratio increased from 0.29 on day 14 to 3.54 on day 16; in lung it rose from 0.29 on day 16 to 6.7 on day 19. Injection of mothers 16 hr earlier with dexamethasone increased the ratio in lung and placenta on day 16 but not earlier. The unchanged ³H-corticosterone recovered from fetal tissues 15 min after injection into mothers was <3% on day 14 but increased manyfold in all tissues by day 19. It is concluded that corticosteroids are regulated in fetal mouse tissues by the interconversion of the hormone and its 11-dehydro metabolite, and that the pattern varies in different tissues and changes with gestational age.     

Iminosugars: potential inhibitors of liver glycogen phosphorylase

The first synthesis of the single isomers (3R,4R,5R); (3S,4S,5S): (3R,4R,5S) and (3S,4S,5R) of 5-hydroxymethyl-piperidine-3,4-diol from Arecolin is reported, including the synthesis of a series of N-substituted derivatives of the (3R,4R,5R)-isomer (Isofagomine). The inhibitory effect of these isomers as well as of a series of N-substituted derivatives of the (3R,4R,5R)-isomer and selected hydroxypiperidine analogues on liver glycogen phosphorylase (GP) showed that the (3R,4R,5R) configuration was essential for obtaining an inhibitory effect at submicromolar concentration. The results also showed that all three hydroxy groups should be present and could not be substituted, nor were extra OH groups allowed if sub-micromolar inhibition should be obtained. Some inhibitory effect was retained for N-substituted derivatives of Isofagomine; however, N-substitution always resulted in a loss of activity compared to the parent compound, IC50 values ranging from 1 to 100 microM were obtained for simple alkyl, arylalkyl and benzoylmethyl substituents. Furthermore, we found that it was not enough to assure inhibitory effect to have the (R,R,R) configuration. Fagomine, the (2R,3R,4R)-2-hydroxymethylpiperidine-3,4-diol analogue, showed an IC50 value of 200 microM compared to 0.7 microM for Isofagomine. In addition, Isofagomine was able to prevent basal and glucagon stimulated glycogen degradation in cultured hepatocytes with IC50 values of 2-3 microM.     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Synthesis of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives as inhibitors of human liver glycogen phosphorylase a

A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide (2f), inhibited hLGPa with an IC(50) of 0.90microM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) of 0.62microM and oral hypoglycemic activity in diabetic db/db mice. Crystallographic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface, with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

An engineered liver glycogen phosphorylase with AMP allosteric activation

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.     

The nucleotide sequence of rat liver glycogen phosphorylase cDNA.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.     

Isolation and study of a liver alpha-amylase. Comparison with glycogen phosphorylase activity

An oligomaltosaccharide-forming amylase has been observed in mice liver crude homogenate. This enzyme has been isolated by binding to amylose. Some of its functional parameters have been studied and compared with those of glycogen phosphorylase demonstrating that amylase activity is not due to a glycogen phosphorylase isoenzyme. It has been further observed that amylase needs Ca2+ of Mg+2 and Cl- for its activity.     

Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase.

The glycogen particle - glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.     

Observations on the histochemically demonstrable liver glycogen phosphorylase activity and native glycogen under different nutritional conditions

Summary In this study a histochemical demonstration of glycogen phosphorylase activity and native glycogen in the livers from normally fed, overfed and starved rats was performed.It was found that the amount and localization of phosphorylase activity well corresponded to the amount and localization of the native glycogen. A change of the glycogen content in the liver also resulted in a change of the histochemically demonstrable liver glycogen phosphorylase activity.It is concluded that the presence of tissue bound glycogen and undissolved glycogenphosphorylase complexes are necessary for positive histochemical demonstration of liver glycogen phosphorylase activity.This work was supported by a grant from the Finnish Veterinary Medical Foundation.     

The effects of isofagomine, a potent glycogen phosphorylase inhibitor, on glycogen metabolism in cultured mouse cortical astrocytes

A novel inhibitor of liver glycogen phosphorylase, isofagomine, was investigated as a possible inhibitor of the enzyme in the brain and in cultured astrocytes. Additionally, the effect of the drug on norepinephrine (NE) induced glycogen degradation in astrocytes was studied. Astrocytes were cultured from mouse cerebral cortex and homogenates were prepared from the cells as well as from mouse brain. Isofagomine dose-dependently inhibited glycogen phosphorylase when measured in the direction of glycogen degradation in both preparations with IC50 values (mean +/- SEM) of 1.0 +/- 0.1 microM and 3.3 +/- 0.5 microM in brain and astrocyte homogenates, respectively. Moreover, isofagomine at a concentration of 400 microM completely prevented NE induced depletion of glycogen stores and the concomitant lactate production in intact astrocytes. It is suggested that this novel glycogen phosphorylase inhibitor may be a valuable tool to investigate the functional importance of glycogen in astrocytes and in the brain.