An assessment of the importance of intralysosomal and of α‐amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen‐storage disease

Abstract

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase.In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a ; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of a-amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate, already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of a-amylase and a-glucosidase.Evidence was obtained that gsd hepatocytes are more fragile, and that amylolysis of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick-frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CC14) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats.The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats.Taken together, the data indicate that only the phosphorolytic degradation of glycogen is metabolically important, and that a-amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.Kinetic data on the two forms of liver phosphorylase indicate that the rate of glycogenolysis in the liver is determined by the concentration of phosphorylase a [l]. However, this relationship has only been directly demonstrated with isolated hepatocytes incubated with or without glucagon [2].

Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.

“…In conclusion, BAY R3401 reduces glycogenolysis, both by inactivation and by inhibition of residual phosphorylase a, which is solely responsible for glycogenolysis in the liver (30). To what extent BAY R3401 will then also promote glycogen deposition depends on several factors: 1) suppression by the inhibitor of futile glycogen cycling driven by phosphorylase a and 2) activation of glycogen synthase.…”

“…When 125 µmol/l BAY R3401 was added, the rate of glycogenolysis dropped precipitously and then slowly declined further during the next 30 min. In the intact liver, glycogenolysis is exclusively catalyzed by phosphorylase a (30,49). The drop in glycogenolytic output (to 17 ± 1% of preinhibitor rates, n = 5) could be explained in part by a drop in phosphorylase a levels (to 37 ± 4% of preinhibitor levels) (Fig.…”

“…Assay of glycogen phosphorylase phosphorylation state. Phosphorylase was assayed in the synthetic direction in a lyotropic buffer with 5% dimethoxymethane and 2 mmol/l AMP for assay of total enzyme or with 0.5 mmol/l caffeine for selective assay of the a-form (30). Selective assay of glycogen phosphorylase a. Phosphorylase a was assayed in the physiologic glycogenolytic direction by a spectrophotometric method (31).…”

“…The glycogen-synthetic phosphorylase assay, in which the release of inorganic phosphate is measured, is commonly used for assay of phosphorylase activity. The test has been optimized for the quantitative determination of the phosphorylation state of phosphorylase (30). We wanted to study the effects of the inhibitor BAY U6751 on the comparative assay of phosphorylase a and b.…”

“…3) of the kinetic behavior of phosphorylase a, from liver and muscle, assayed in the physiologic degradative direction under physiologic conditions in which only phosphorylase a is active (39). In liver, the a-form is solely responsible for glycogenolysis (30). Both the muscle and liver enzymes are sensitive to BAY U6751 and, at least for the muscle enzyme, inhibition is overcome by addition of AMP.…”