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PubMed2006

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats.

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

Animals↗
PubMed1976

Metabolism of the reserve polysaccharide of Streptococcus mitior (mitis): is there a second alpha-1,4-glucan phosphorylase?

The alpha-1,4-glucan phosphorylase (alpha-1,4-glucan: orthophosphate glucosyltransferase; EC 2.4.1.1) associated with the particulate cell fraction of Streptococcus mitior strain S3 was compared with the soluble maltodextrin phosphorylase that had been previously isolated from the same organism (Walker et al., 1969). The particulate enzyme was more sensitive to the glycogen content of the cell than the soluble euzyme; its activity was highest when the cells were grown under conditions favoring high glycogen storage. Substrate specificities of the two high activity towards endogenous glycogen, whereas low-molecular-weight maltodextrins were the preferred substrates for the soluble phosphorylase. The purification of the particulate phosphorylase included incubation of the particulate fraction in 160 mM sodium phosphate-10 mM sodium citrate-0.1% (wt/vol) Triton X-100 buffer (pH 6.7) and ion-exchange chromatography on diethylamino-ethyl- Sephadex A-50. The purified enzyme was fully soluble. The value for the purification factor was variable and depended on (i) the substrate used and (ii) whether the synthetic or the degradative reaction was being measured. The solubilization resulted in considerable changes in the properties of the phosphorylase: the pH optimum for activity was raised from 6.0 to 7.0-7.5 and the substrate specificity was altered. Consequently, the purified enzyme bore greater similarity to the soluble maltodextrin phosphorylase. The reported results are best explained in terms of a single phosphorylase, the specificity which is determind by its binding state in the cell. The enzyme acts as a glycogen phosphorylase in the particulate state and as a maltodextrin phosphorylase when soluble. The equilibrium between the two forms is related to the glycogen content of the cells.

Glycogen↗
PubMed1993

Glycogen phosphorylase: developmental expression in rat liver.

The developing fetus is vulnerable to hypoglycemia if its transplacental substrate supply is compromised. Consequently, we examined hepatic glycogen phosphorylase gene expression in the developing rat liver by comparing the relative activities of the hepatic phosphorylase enzyme, concentrations of cellular phosphorylase mRNA, and rates of transcription in isolated liver nuclei of fetal rats at 19 days of gestation, 21 days of gestation, neonatal rats, and suckling rats. Cellular phosphorylase mRNA and enzyme activity (total and phosphorylase a) increased until term. Reciprocally, the rate of phosphorylase mRNA transcription was rapid in the rats at 19 days of gestation, and declined progressively until term. These data indicate that glycogen phosphorylase gene expression is regulated post-transcriptionally in late gestation, perhaps by an increase in phosphorylase mRNA stability towards term. This results in increased phosphorylase mRNA and enzyme expression.

Aging↗
PubMed2005

Increased potency and efficacy of combined phosphorylase inactivation and glucokinase activation in control of hepatocyte glycogen metabolism.

Glucokinase and phosphorylase both have a high control strength over hepatocyte glycogen metabolism and are potential therapeutic targets for type 2 diabetes. We tested whether combined phosphorylase inactivation and glucokinase activation is a more effective strategy for controlling hepatic glycogen metabolism than single-site targeting. Activation of glucokinase by enzyme overexpression combined with selective dephosphorylation of phosphorylase-a by an indole carboxamide that favors the T conformation of phosphorylase caused a greater stimulation of glycogen synthesis than the sum of either treatment alone. This result is explained by the complementary roles of elevated glucose-6-phosphate (G6P; a positive modulator) and depleted phosphorylase-a (a negative modulator) in activating glycogen synthase and also by synergistic inactivation of phosphorylase-a by glucokinase activation and the indole carboxamide. Inactivation of phosphorylase-a by the indole carboxamide was counteracted by 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside, which is metabolized to an AMP analog; this effect was reversed by G6P. Our findings provide further evidence for the inverse roles of G6P and AMP in regulating the activation state of hepatic phosphorylase. It is proposed that dual targeting of glucokinase and phosphorylase-a enables improved potency and efficacy in controlling hepatic glucose metabolism.

Adenosine Monophosphate↗
PubMed1989

The family of glycogen phosphorylases: structure and function.

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.

Amino Acid Sequence↗
PubMed1975

Regulation of glycogen phosphorylase. Role of the peptide region surrounding the phosphoserine residue in determining enzyme properties.

A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.

Adenosine Monophosphate↗
PubMed2002

Role of thymidine phosphorylase in an in vitro model of human bladder cancer invasion.

PURPOSE: It has been previously demonstrated that the angiogenic factor thymidine phosphorylase is elevated significantly in invasive bladder cancer. We report that it is not merely an incidental finding. Thymidine phosphorylase has a functional role in bladder cancer invasion. MATERIALS AND METHODS: The superficial bladder cancer cell line RT112 was transfected by retroviral techniques to generate the RT112-TP clone that expressed significantly elevated levels of thymidine phosphorylase, comparable to those of invasive human bladder cancers. The empty vector control RT112-EV was generated for comparison. Growth of these transfectants was examined using a new in vitro model of bladder cancer invasion based on de-epithelialized rat bladder and by assessing growth as xenografts in nude mice. The effect of 5-deoxy-5-fluorouridine, a prodrug activated by TP to produce 5-fluorouracil, was also examined. RESULTS: RT112-TP high thymidine phosphorylase expressing cells invaded into the stroma of the in vitro model but wild-type RT112 and RT112-EV cells did not. This invasion was abolished by 5-deoxy-5-fluorouridine. Invasion correlated with thymidine phosphorylase expression on immunohistochemical testing. There was also a significantly greater xenograft growth rate for RT112-TP than for RT112-EV, confirming the malignant growth advantage conferred by thymidine phosphorylase. CONCLUSIONS: We demonstrated that thymidine phosphorylase may have a functional role in bladder cancer invasion and the apparent advantage of thymidine phosphorylase expression to tumor cells can be exploited by therapies that utilize prodrugs such as 5-deoxy-5-fluorouridine, which is activated by thymidine phosphorylase and inhibited invasion in our model.

Animals↗
PubMed1992

Coordinated expression of phosphorylase kinase subunits in regenerating skeletal muscle.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

Amino Acid Sequence↗
PubMed1989

Effects of pyrimidine nucleoside phosphorylase inhibitors on hepatic fluoropyrimidine elimination in the rat.

The breakdown of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) is catalyzed by the pyrimidine nucleoside phosphorylases, uridine phosphorylase and thymidine phosphorylase. The effects of nucleoside phosphorylase inhibitors on FdUrd and FUra elimination by the isolated perfused rat liver were investigated. The inhibitor was injected into the perfusion reservoir 5 min before FdUrd or FUra, and serial perfusion fluid samples were collected for fluoropyrimidine analysis. The disappearance of each fluoropyrimidine followed Michaelis-Menten kinetics, as shown previously. 6-Benzyl-2-thiouracil, a thymidine phosphorylase-selective inhibitor, and 1-(2'-deoxy-beta-D-glucopyranosyl)thymine, a uridine phosphorylase-selective inhibitor, each decreased the rate of FdUrd disappearance (apparent Ki, 1.4-1.6 and 3.8 mM, respectively) but had no direct effect on FUra disappearance. However, 6-benzyl-2-thiouracil decreased the peak concentration of FUra derived from administered FdUrd and increased the t 1/2 of disappearance of derived FUra due to its delayed formation. 2,6-Dihydroxypyridine, a uridine phosphorylase-selective inhibitor, decreased the rate of FdUrd disappearance (apparent Ki, 12.4-16.2 microM) and directly inhibited FUra elimination (apparent Ki, 4.3-5.3 microM). 2,4-Dihydroxypyridine, which does not inhibit pyrimidine nucleoside phosphorylases, directly inhibited FUra elimination (apparent Ki, 77 microM) and also decreased the rate of FdUrd disappearance, possibly due to product (FUra) inhibition. It was concluded that the hepatic elimination of FdUrd is slowed by pyrimidine nucleoside phosphorylase inhibitors and that some of these drugs block FUra, as well as FdUrd, elimination.

Animals↗
PubMed1993

Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin.

Neuronal tissue-specific proteins B-50 (GAP-43, neuromodulin) and neurogranin are phosphorylated by phosphorylase kinase with stoichiometries of 0.4 and 0.5 mol of phosphate/mol of protein, respectively. The apparent Km and kcat values determined at pH 8.2 for neurogranin phosphorylation are 28.4 microM and 139.3 min-1, respectively, and for B-50 phosphorylation are 22.8 microM and 33.2 min-1, respectively. As a substrate of phosphorylase kinase, phosphorylase is approximately 44 and approximately 13 times better than B-50 and neurogranin, respectively. Both proteins are better substrates of protein kinase C than of phosphorylase kinase and are phosphorylated on a single site by phosphorylase kinase. The sequence analyses of tryptic phosphopeptides isolated from neurogranin and B-50 phosphorylated by phosphorylase kinase revealed the same amino acid sequence, IQASF, indicating that phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of B-50 and neurogranin previously known to be phosphorylated by protein kinase C (Coggins, P. J., and Zwiers, H. (1989) J. Neurochem. 53, 1895-1901; Baudier, J., Deloulme, J. C., Dorsselaer, A. V., Black, D., and Matthes, W. D. (1991) J. Biol. Chem. 266, 229-237). In rat brain synaptosomes, a relatively high phosphorylase kinase specific activity is detected, and approximately 32% activity is associated with synaptic membranes where B-50 is localized. In rat brain homogenate and synaptosomal membranes, phosphorylation of a protein that co-migrates with B-50 on SDS-polyacrylamide gel electrophoresis is enhanced in the presence of exogenous phosphorylase kinase.

Amino Acid Sequence↗
PubMed1976

A study on the autoactivation of rabbit muscle phosphorylase kinase.

Under conditions favoring its autocatalytic reaction, phosphorylase kinase may be activated and phosphorylated in 2-(N-morpholino)ethanesulfonate (Mes) buffer to a much higher level than in beta-glycerophosphate buffer. The fact that the reaction is autocatalytic is supported by several observations: (a) the progress curve of the reaction exhibits a pronounced lag phase, (b) the reaction is strongly inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetate, which inhibits phosphorylase kinase, (c) the pH profile of the reaction resembles that of the phosphorylase b to a reaction as catalyzed by nonactivated phosphorylase kinase, and (d) the reaction is not significantly affected by adenosine 3':5'-monophosphate (cAMP) nor by the heat-stable protein inhibitor of cAMP-dependent protein kinases. When fully autoactivated, phosphorylase kinase possesses an activity that is 100% higher than that of the protein kinase-activated form. The results suggest that autophosphorylation of phosphorylase kinase may be an important regulatory mechanism. The autocatalytic reaction involves phosphorylation of the two larger subunits of phosphorylase kinase, i.e. subunits A and B, with a combined total of 7 to 9 phosphates incorporated per mol of enzyme. Although the cAMP-dependent protein kinase also catalyzes the phosphorylation of subunits A and B, the two mechanisms of phosphorylation appear to involve different sites. Prior phosphorylation of phosphorylase kinase by the protein kinase has little effect on the level of autophosphorylation. Thus activation of phosphorylase kinase may be brought about by phosphorylation of the enzyme at different sites.

Adenosine Triphosphate↗
PubMed1980

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP.

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.

Adenosine Monophosphate↗
PubMed1983

Limited proteolysis by subtilisin reveals structural differences between phosphorylase a and b.

The limited proteolysis of rabbit skeletal muscle phosphorylase a and b was studied with subtilisin BPN' immoblized to Sepharose 4B. The activity of phosphorylase b is nearly resistant to subtilisin under the conditions (pH 7.0, 30 degrees C) where phosphorylase a rapidly loses its activity. The pH profile of phosphorylase a and b digestion is different. Proteolytic fragments of mol. wt 70,000 and 30,000 generated from phosphorylase a, while mol. wt 80,000 and 70,000 generated from phosphorylase b can be detected by SDS gel electrophoresis. Addition of AMP to phosphorylase b favours a conformation similar to--but not identical with--phosphorylase a as recognised by subtilisin action.

Adenosine Monophosphate↗
PubMed1982

Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases.

Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms.

Escherichia coli↗
PubMed2001

Glycogen phosphorylase reactivity in the amygdala and bed nucleus of the stria terminalis.

The present study examines the reactivity of the glial metabolic enzyme, glycogen phosphorylase, within the amygdala and bed nucleus of the stria terminalis. Reactivity for phosphorylase a, the active form of glycogen phosphorylase, was higher in all parts of the medial amygdaloid nucleus, in the medial division of the central amygdaloid nucleus, in the anterior amygdaloid area and in the bed nucleus of stria terminalis than in all parts of the lateral amygdaloid nucleus, the anterior cortical amygdaloid nucleus, the posteromedial and posterolateral cortical amygdaloid nuclei, the intercalated nucleus of the amygdala, main part and the intercalated nuclei. A greater degree of phosphorylase a reactivity was also observed in the basolateral amygdaloid nucleus, anterior and posterior parts, and in the basomedial amygdaloid nucleus, anterior part, while other parts of these nuclei were less reactive. Reactivity attributed to total glycogen phosphorylase enzyme, phosphorylase a+phosphorylase b activated by AMP, was higher and homogeneous across the amygdala. Phosphorylase a patterns are likely to reflect differences in the contribution of glycogenolysis to the metabolic support of cells in the amygdala and bed nucleus of the stria terminalis. Possible relationships to local neuronal activity and to differences in glycogenolytic neuromodulatory input are discussed.

Amygdala↗
PubMed1981

Use of arginine compounds to examine the role of an essential arginine in the mechanism of glycogen phosphorylase.

The possible role of arginine in the mechanism of muscle glycogen phosphorylase qas studied by examining the effect of arginine compounds. Guanidino compounds with an aromatic group inhibit native phosphorylase b, reduced phosphorylase b, phosphorylase b', and phosphorylase a. The inhibition was found to be uncompetitive with respect to glucose 1-phosphate and noncompetitive toward glycogen for phosphorylase b. This is consistent with a kinetic mechanism where the inhibitor binds after the substrate, glucose 1-phosphate. In the presence of citrate and l-cysteine, N-alpha-tosylarginine methyl ester, a good inhibitor, promotes the removal of tightly bound pyridoxal phosphate. Potato phosphorylase has many similarities to the muscle enzyme, but it lacks the regulatory sites and does not have a polysaccharide storage site [Shimomura, S., & Fukui, T. (1980) Biochemistry 19, 2287]. N-alpha-Tosylarginine methyl ester inhibited the potato enzyme, was uncompetitive with glucose 1 -phosphate, and was competitive with starch; therefore, it seems likely that TAME is binding near the active site of both the potato and muscle enzyme. The different inhibitory patterns with respect of polysaccharide for potato and muscle phosphorylase can be explained by the absence of the polysaccharide storage site on the potato enzyme. Inhibition by arginine compounds is related to the pKa of the guanidino fuction, i.e., the lower pKa value, the greater the inhibition. On the basis of these studies and those of Dreyfus et al. [Dreyfus, M., Vandenbunder, B., & Buc, H. (1980) Biochemistry 19, 3834], who found that Arg-568 was essential for activity, we suggest that arginyl compounds inhibit when an unprotonated guanidino group competes for the "binding site" of Arg-568.

Animals↗
PubMed1990

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms.

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

Anesthesia, General↗
PubMed1979

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.

Adrenalectomy↗