Nigel J. Pearce

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

BACKGROUND Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Accelerated atherosclerosis in C57Bl/6 mice transplanted with ApoE-deficient bone marrow

Apolipoprotein E (apoE), a high affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage cell lineage. The role of monocyte/macrophage-derived apoE in atherogenesis was assessed by transplantation of apoE-deficient (apoE-/-) bone marrow into normolipidemic C57Bl/6 mice. No significant effect could be demonstrated on serum apoE levels in C57Bl/6 mice, transplanted with apoE-deficient bone marrow compared with control transplanted mice. Furthermore, no consistent effect on serum cholesteryl esters and triglyceride concentrations could be demonstrated on either a standard chow diet or a high cholesterol diet. Quantitative analysis of atherosclerosis in mice transplanted with apoE-deficient bone marrow, after two months on a high cholesterol diet, revealed a 4-fold increase in the atherosclerotic lesion area as compared to animals transplanted with apoE+/+ bone marrow. Analysis of the ability of apoE-deficient macrophages to release cholesterol after loading with acetylated LDL revealed that the release of cholesterol from apoE-deficient macrophages was impaired as compared to wild-type macrophages in the absence and the presence of specific cholesterol acceptors. In conclusion, apoE production by macrophages retards the formation of atherosclerotic plaques, possibly by mediating cholesterol efflux. We anticipate that pharmacological approaches to increase apoE synthesis and/or secretion by macrophages might be beneficial for the treatment of atherosclerosis.

Effects of cholestyramine on lipoprotein levels and metabolism in Syrian hamsters

Oral administration of cholestyramine to adult male hamsters not only induced a marked decrease in plasma concentrations of cholesterol and LDL but had a similar lowering effect on plasma triacyglycerol and VLDL concentrations. The hypotriglyceridaemic effects of resin administration were not due to an increase in lipoprotein lipase, as post-heparin plasma lipoprotein lipase activities were unchanged, but rather to a 35% decrease in VLDL synthesis. Measurement of the disappearance rate of apolipoprotein B from VLDL after i.v. injection of 125I-labelled hamster or human VLDL into control and cholestyramine-fed recipient animals showed a 2-times lower T1/2 in the drug-treated animals. The fraction of VLDL apolipoprotein B, recovered at any time after injection in the LDL, was equal or higher in cholestyramine-fed animals as compared to controls. These data indicate that the lowering in plasma LDL by cholestyramine in male hamsters is due not only to LDL receptor up-regulation but also to a lower rate of VLDL synthesis. No indications were found for a decreased efficiency of VLDL to LDL conversion in cholestyramine-fed animals.

Bile acid synthesis in hamster hepatocytes in primary culture: Sources of cholesterol and comparison with other species

The synthesis of bile acids by primary hamster hepatocytes in culture has been studied. Measurable rates of bile acid synthesis were obtained from cells prepared from livers of animals fed 2% w/w cholestyramine to induce the synthesis of bile acids through the rate-limiting enzyme cholesterol 7 alpha-hydroxylase. The effects of various sources of substrate for bile acid synthesis in these cultured cells were examined over a period of 24 h and the results compared with published or parallel studies in primary rat hepatocytes or in the human hepatoma cell line, HepG2. In all the cells, bile acid synthesis was stimulated by the addition of 7 alpha-hydroxycholesterol, indicating the rate-limiting role of the cholesterol 7 alpha-hydroxylase. Bile acid synthesis in the hamster hepatocytes was also stimulated by a variety of sources of cholesterol as substrate, mevalonic acid (increasing the production of newly-synthesised cholesterol in the cell), and as an exogenous source, hamster LDL. Similarly, if cholesterol was diverted from intracellular esterification using the ACAT inhibitor Dup128, a further increase in bile acid synthesis could be demonstrated. These results show that hepatocytes obtained from cholestyramine-treated hamsters are deficient in substrate cholesterol for bile acid synthesis. A similar conclusion can be drawn from the published work with rat hepatocytes and is further supported by experiments on the regulation of cholesterol 7 alpha-hydroxylase activity at the mRNA and the protein level, although some in vivo studies in animals and studies in man have led authors to suggest that cholesterol 7 alpha-hydroxylase is saturated with substrate.