SummaryBackground: Patients with type 2 diabetes have a 2-4 fold increased risk of mortality from cardiovascular disease compared to age-matched non-diabetic subjects. This is mainly due to premature atherosclerosis which in part can be accounted for by hyperglycaemia. Early events in human atherosclerosis occur in the intima of the vascular epithelia. Macrophages are the most prominent cell type in atherosclerotic lesions, and uptake of oxidised LDL and accumulation of cholesterol esters in monocyte/macrophages (foam cell formation) is an important early event in atherosclerosis. Previous work in J774.2 murine macrophages exposed to hyperglycaemia in combination with insulin or leptin, show that the rate of cholesterol ester deposition significantly increased through down-regulation of hormone-sensitive lipase (HSL) and up-regulation of acyl-CoA-cholesterol acyltransferase (ACAT). The transcriptional repressor Id2 has the capability to suppress HSL promoter activity, and in J774.2 macrophages Id2 is up-regulated by hyperglycaemia via the hexosamine signalling pathway, involving the rate-limiting enzyme, GFAT, and the enzyme responsible for O-linked glycosylation of proteins, OGT, indicating a direct role of Id2 in macrophage foam cell formation. The role of hyperglycaemia and the hexosamine signalling pathway in primary human macrophages has never been established.Methods: Peripheral blood mononuclear cells (PBMC) from healthy donors were differentiated for 7 days into macrophages. The macrophages were cultured for different times under normoglycaemic (5 mM) or hyperglycaemic conditions (20 mM) or in the absence or presence of glucosamine, PUGNAc or fructose at 5 mM glucose. Cells were lysed in RIPA buffer and protein samples were separated by SDS-PAGE. GFAT, OGT, Id1 and Id2 protein expression as well as abundance of O-GlcNAcylated proteins were determined by Western blotting, and subcellular localization of OGT, O-GlcNAcylation and Id2 was studied by immunoflouresence.Results: Fructose and glucosamine induce Id1 and Id2 protein expression as well as the abundance of O-GlcNAclated proteins in primary human macrophages. Moreover, in accordance with previous observations in the J774.2 murine macrophages cell line, Id2 was found to be a target for O-linked glycosylation in primary human macrophages. Furthermore, we demonstrated nucleo-cytoplasmic shuttling of Id2 and OGT by high glucose, but in contrast to observations in J774.2 cells, hyperglycaemia has no effect on the protein levels of Id1 and Id2. Finally, hyperglycaemia did not regulate GFAT and OGT protein levels in primary human macrophages.Conclusion: In resting human macrophages, the hexosamine signalling pathway is involved in the up-regulation of Id1 and Id2 protein, but hyperglycaemia is not able to increase flux through the hexosamine signalling pathway as compared to murine macrophages and does not regulate GFAT and OGT protein levels.