The role of the hexosamine biosynthesis pathway in control of hepatic glucose metabolism

Abstract

Type 2 diabetes is associated with increased hepatic lipogenesis and glucose production. Enzymes of lipogenesis are co-ordinately induced by insulin and glucose. However, the enzyme glucose 6-phosphatase (G6Pc), which catalyses the final reaction in hepatic glucose production is repressed by insulin but induced by glucose and is markedly elevated in type 2 diabetes. Another gene that is repressed by insulin and induced by glucose in muscle is thioredoxin interacting protein (TXNIP), which is abnormally elevated in muscle in type 2 diabetes. TXNIP gene regulation in liver has not been reported. The induction of hepatic lipogenic enzymes by glucose is attributed to the transcription factor ChREBP-Mlx, whereas the glucose-induction of G6Pc is attributed to covalent modification of FOXO transcription factors by O- GlcNAc formed by the hexosamine biosynthesis pathway (HBP). The aim of this thesis was to investigate the role of the HBP in regulation by glucose of G6Pc and TXNIP gene expression in hepatocytes. This thesis investigated three commonly used methods to modulate HBP flux and covalent modification by O-GlcNAc. (1) An inhibitor of glutamine:fructose 6- phosphate amidotransferase (6-diazo-5-oxo-l-norleucine, DON), the rate limiting enzyme of the HBP, was established to be a valid tool to study glucose-regulated gene expression. (2) Substrates that enter the HBP after GFAT, such as glucosamine which is widely used to demonstrate links between HBP or O-GlcNAc modification and insulin resistance were shown to be invalid tools. (3) Inhibitors of O-GlcNAc modification or expression of O-GlcNAc transferase were of limited use to alter protein modification by O-GlcNAc. Glucose caused a larger induction of G6Pc and TXNIP mRNA in the absence of insulin than in its presence, and this induction could be largely accounted for by Mlx- dependent mechanisms (supporting involvement of ChREBP or MondoA) and by FOXO transcription factors. G6Pc and TXNIP expression were confirmed to be regulated by distinct mechanisms based on the induction of TXNIP but not G6Pc by the glucose analogue, 2-deoxyglucose, through an Mlx-independent mechanism. Insulin caused rapid translocation of both FOXO1 and FOXO3A from the nucleus to the cytoplasm. Both glucose and 2-deoxyglucose opposed the translocation of FOXO1 and FOXO3A by insulin, and they stimulated translocation of FOXO3A to the nucleus in the absence of insulin. Inhibition of HBP flux with the GFAT inhibitor had the following effects: (i) counteraction of the glucose-induction of both G6Pc and TXNIP mRNA; (ii) counteraction of glucose-induced translocation of ChREBP to the nucleus without affecting the signalling metabolite, fructose 2,6-bisphosphate; (iii) counteraction of glucose-induced translocation of FOXO1 and FOXO3A to the nucleus. A role for O- GlcNAc modification of both ChREBP and FOXO3A was supported by wheat-germ agglutinin precipitation. The results of this thesis support involvement of both HBP flux and O-GlcNAc modification of ChREBP and FOXO3A in glucose-regulated expression of G6Pc and FOXO3A but they do not support a role for glucosamine as an experimental tool to study glucose-induced insulin resistance.