Rat Liver Glycogen Phosphorylase Research Articles

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.

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver.

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35-1.0 micron) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 microns. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.

Characterization of the 5' flanking region of the gene encoding rat liver glycogen phosphorylase

A genomic region encompassing 800 bp of the promoter-regulatory region and exon 1 of the gene ( LGP) encoding rat liver glycogen phosphorylase has been isolated and characterized. Transcripts of the LGP gene initiate predominantly within an 8-bp region 48-bp upstream from the start codon. Additional transcripts were detected that initiate as far as 95 bp upstream from the start codon. To identify cis-acting sequences involved in regulating transcription, HepG2 cells were transfected with vectors containing serial deletions of the promoter-regulatory region of LGP ligated to the cat reporter gene. Two upstream regions were found to enhance transcription. One of these regions contains an alternating purine-pyrimidine sequence. LGP, which lacks a consensus TATA sequence, is like TATA-less and CAAT-less housekeeping genes in that it contains G + C-rich domains upstream from multiple transcription start points. Nuclear proteins from adult rat tissues bound in a tissue-specific fashion to one of these G + C-rich regions.

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.

The gene encoding rat liver glycogen phosphorylase contains multiple polyadenylation signal sequences

RNA blot analysis of rat liver and adipose tissues detected two glycogen phosphorylase (GP)-encoding transcripts. The polymerase chain reaction was used to characterize the 3′-noncoding region of the gene ( L-GP) encoding liver-GP (L-GP) from the lean Zucker rat ( Fa/Fa). Three distinct classes of colinear cDNA clones were identified by nucleotide (nt) sequence analysis, demonstrating that the L-GP gene contains at least three functional polyadenylation sites. The predominant L-GP transcript was generated by polyadenylation 130 nt 3′ from the end of the coding region. A previously uncharacterized L-GP transcript is generated by polyadenylation at 346 nt 3′ of the first polyadenylation site. Polyadenylation site selection does not appear to be regulated in a tissue-specific fashion. The relative steady-state L-GP mRNA levels in the different types of adipose tissues were comparable to, or exceeded transcript levels in liver.

Regulation of rat liver glycogen phosphorylase concentration by in vivo relative levels of glucagon and insulin.

The concentrations of glycogen phosphorylase protein were determined by rocket immunoelectrophoresis in liver extracts from rats that had artificially induced altered hormonal patterns. These levels were compared with measurements of total phosphorylase activity. Minipump-induced chronic hyperglucagonemia and streptozotocin-induced diabetes resulted in 47% and 67% decreases, respectively, in total phosphorylase activity along with corresponding 52% and 68% drop, respectively, in phosphorylase protein levels. Insulin replacement in diabetic rats returned both parameters to control values. Minipump-induced hyperinsulinemia or injection of glucagon antiserum, T3, or propylthiouracil had no effect. The results of this study indicate that conditions which lead to an elevation of the glucagon to insulin molar ratio to values higher than 1.0 cause a significant decrease in the liver phosphorylase protein level.

Effects of chronic ethanol ingestion on male and female rat liver glycogen phosphorylase phosphatase.

The effects of chronic ethanol ingestion on the interconversion of the active to the inactive form of glycogen phosphorylase by phosphorylase phosphatase was studied. Male and female rats also were compared. Chronic ethanol feeding decreased phosphorylase a and total phosphorylase activity in male rats. In females, no change was observed in phosphorylase a, whereas total phosphorylase activity was increased 73%. This was found to correlate with the relative activities of phosphorylase phosphatase. The data show differences between the two sexes with regard to the AMP inhibition of phosphorylase phosphatase and the caffeine stimulation of the phosphatase. Ethanol markedly enhanced the AMP inhibition of the phosphatase in males but had no effect in females. Further studies in females showed that ethanol completely obliterated the well documented stimulation of the phosphatase by caffeine; however, it did not alter the caffeine effect in males. These data suggest possible alterations in the tertiary structure of phosphorylase a.

Inhibition of gluconeogenesis and glycogenolysis by 2,5-anhydro-D-mannitol.

2,5-Anhydro-D-mannitol (100 to 200 mg/kg) decreased blood glucose by 17 to 58% in fasting mice, rats, streptozotocin-diabetic mice, and genetically diabetic db/db mice. Serum lactate in rats was elevated 56% by 2,5-anhydro-D-mannitol, but this could be prevented by dichloroacetate (200 mg/kg) or thiamin (200 mg/kg). In hepatocytes from fasted rats, 1 mM 2,5-anhydro-D-mannitol inhibited gluconeogenesis from a mixture of alanine, lactate, and pyruvate. It also inhibited glucose production and stimulated lactate formation from glycerol or dihydroxyacetone. Glycogenolysis in hepatocytes from fed rats was markedly inhibited by 1 mM 2,5-anhydro-D-mannitol both in the presence or absence of 1 microM glucagon. 2,5-Anhydro-D-mannitol can be phosphorylated by fructokinase or hexokinase to the 1-phosphate and then by phosphofructokinase to the 1,6-bisphosphate. Rat liver glycogen phosphorylase was inhibited by 2,5-anhydro-D-mannitol 1-phosphate (apparent Ki = 0.66 +/- 0.09 mM) but was little affected by 2,5-anhydro-D-mannitol 1,6-bisphosphate. Rat liver phosphoglucomutase was inhibited by 2,5-anhydro-D-mannitol 1-phosphate (apparent Ki = 2.8 +/- 0.2 mM), whereas 2,5-anhydro-D-mannitol 1,6-bisphosphate served as an alternative activator (apparent K alpha = 7.0 +/- 0.5 microM). Rabbit liver pyruvate kinase was activated by 2,5-anhydro-D-mannitol 1,6-bisphosphate (apparent K alpha = 9.5 +/- 0.9 microM), whereas rabbit liver fructose 1,6-bisphosphatase was inhibited by 2,5-anhydro-D-mannitol 1,6-bisphosphate (apparent Ki = 3.6 +/- 0.3 microM). The phosphate esters of 2,5-anhydro-D-mannitol would, therefore, be expected to inhibit glycogenolysis and gluconeogenesis and stimulate glycolysis in liver.

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

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

Alteration of hepatic glycogen synthase phosphatase activity by insulin deficiency

Perfused livers from normal and alloxan-diabetic rats were studied to determine whether the diabetes-related decrease in glycogen synthase phosphatase was due to an alteration of the synthase molecule, an increase in synthase phosphatase activity inhibition by phosphorylase a, or generation of inhibitor of the phosphatase. With purified rat liver synthase as substrate for the phosphatase, the diabetic tissue remained 90-95% deficient in the ability to catalyze synthase D to I conversion, showing that the defect cannot be solely due to an altered substrate. When synthase phosphatase assays were carried out in the presence of rat liver glycogen phosphorylase antiserum, phosphatase activity remained 70-75% deficient in diabetic tissue. Therefore, the defect cannot be attributed to increased inhibition of synthase phosphatase by increased amounts of phosphorylase a. When synthase phosphatase assays were run by mixing extracts from normal and diabetic livers, phosphatase activity was additive, indicating that a phosphatase inhibitor was probably not involved in the phosphatase deficiency in the diabetic. These data are consistent with the hypothesis that the diabetes-related defect in glucose regulation of hepatic glycogen synthase is due to a molecular alteration or a deficiency of a specific glycogen synthase phosphatase.

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

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

Lead and some other metals in the histochemical demonstration of rat liver glycogen phosphorylase activity.

Lead in a concentration of 0.25 mM was tried as a histochemical trapping agent for inorganic phosphate, liberated in the reversible reaction catalyzed by the liver glycogen phosphorylase.

Histochemical demonstration of rat liver glycogen phosphorylase activity with iron (Fe++).

A new histochemical method for light microscopic demonstration of liver glycogen phosphorylase activity has been introduced in this study.