Expression of Glutamine:Fructose-6-Phosphate Amidotransferase in Human Tissues: Evidence for High Variability and Distinct Regulation in Diabetes

Abstract

Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucose-induced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated.

Molecular Mechanisms of Diabetic Retinopathy, General Preventive Strategies, and Novel Therapeutic Targets

“…UDP-GlcNAc is a substrate for O-linked glycosylation, which is catalyzed by O-GlcNAc transferase. UDP-GlcNAc, the major product, is the unique donor for the O-linkage of a single N-acetylglucosamine molecule (O-GlcNAc) to many cytoplasmic and nuclear proteins [51, 52]. Glucose is rapidly phosphorylated to glucose-6-phosphate after entering the cell which can then oxidize via glycolysis or the pentose phosphate shunt or stored as glycogen.…”

“…Clinically, the increased GFAT activity has been well interrelated with HbA1c levels in diabetic patients [59]. Distinct and high expression of GFAT was also demonstrated in diabetic nephropathy and other complications [52]. Despite a number of studies and data documentation saying that most of the tissues express GFAT, in eye specific tissues the data on GFAT is still lacking [51].…”

Role of Endoplasmic Reticulum Stress in Atherosclerosis and Diabetic Macrovascular Complications

“…In the hexosamine pathway, glutamine: fructose-6 phosphate amidotransferase (GFAT) catalyzes conversion of glucose to glucosamine-6 phosphate (G-6P) [106]. Diabetic hyperglycemia activates the hexosamine pathway that leads to the production of elevated G-6P levels in vascular cells [107, 108].…”

The Role of Glucosamine-Induced ER Stress in Diabetic Atherogenesis

“…When intracellular glucose levels rise, flux through this pathway increases. Furthermore, increased GFAT expression and activity have been reported in tissues from humans with diabetes [48]. The net result is an elevated intracellular concentration of glucosamine.…”

“…Recently, small clinically relevant studies involving humans with metabolic syndrome have been carried out. Patients with diabetic nephropathy have been shown to have increased GFAT expression in glomerular epithelial and mesangial cells and that GFAT is expressed in most tissues involved in diabetic complications [48, 117]. Pancreatic beta cells isolated from type 2 diabetics have been shown to have marked expression of ER stress markers [118] and increased susceptibility to ER stress compared to nondiabetic controls [119] and that ER stress may contribute to IL-1 β production, mild islet inflammation [120], and β -cell failure [118].…”