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Prostaglandins E2 and F2 alpha affect glycogen synthase and phosphorylase in isolated hepatocytes.

ABSTRACT: Prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) inactivated glycogen synthase and activated glycogen phosphorylase in rat hepatocytes in a dose- and time-dependent manner. These effects were dependent on the presence of Ca2+ in the incubation medium. When glycogen synthase was immunoprecipitated from cells incubated with [32P]Pi and then treated with PGE2 or PGF2 alpha, there was increased phosphorylation of the 88 kDa subunit of the enzyme. This phosphorylation affected two CNBr fragments of the glycogen synthase, CB-1 and CB-2, the same fragments that are phosphorylated by different glycogenolytic hormones. No phosphorylation of glycogen synthase by prostaglandins was observed in the absence of Ca2+. Thus the effect of PGE2 and PGF2 alpha on these glycogen-metabolizing enzymes supports a role for regulation by prostaglandins of glucose metabolism in parenchymal liver cells.

SUBMITTER: Gomez-Foix AM

PROVIDER: S-EPMC1138786 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Project description:A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8 mM 1-GlcNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced the dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold improvement on glucose for the liver activity and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP inactivation but failed to elicit activation of GS. Glucose-induced activation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosine analogue and kinase inhibitor, 5'-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylation of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cells at 20 mM 1-GlcNAc but was reduced by the presence of 50 microM ITU (0.775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure of 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.

Project description:Background and purposeGlycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells.Experimental approachThe effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes.Key resultsGPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice.Conclusion and implicationsThese data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes.Linked articlesThis article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

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Prostaglandins E2 and F2 alpha affect glycogen synthase and phosphorylase in isolated hepatocytes.

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