Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. It has been established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesized that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. In support of the hypothesis we found that glucosamine-supplemented apoE-/- mice had elevated levels of ER stress and atherosclerosis. Mechanistically, our data showed that glucosamine induced ER stress by interfering with the lipid-linked oligosaccharide biosynthesis pathway and protein N-glycosylation. These findings support a model by which conditions of hyperglycemia promote vascular complications through a glucosamine-intermediate. / Thesis / Doctor of Philosophy (PhD) / Diabetes mellitus dramatically increases the risk for heart attacks and strokes. High blood glucose is utilized in cells through its conversion into metabolites, such as glucosamine. We hypothesized that conditions of high blood glucose can led to an increase in intracellular glucosamine which can initiate pathways involved in accelerating atherosclerosis. Our results show that this is possible in both human cells and mice.

<p>Human L-glutamine:D-fructose-6-phosphate amidotransferase 1 (hGFAT1) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is a potential target to help prevent secondary complications of type II diabetes. GFAT catalyzes the irreversible reaction between L-glutamine and D-fructose-6-phosphate to produce L-glutamate and D-glucosamine-6-phosphate. hGFAT1 is not commercially available and is difficult to obtain from natural sources. Thus, a recombinant method to generate and purify the enzyme was developed and is discussed herein.</p> <p>There are only a handful of known inhibitors available to study the enzyme and the majority of these are toxic, non-specific, or substrate analogs. A high-throughput screening campaign was undertaken in pursuit of novel hGFAT1 inhibitors. The bioactive subset of the Canadian Compound Collection was assayed in duplicate for GFAT inhibitory activity using a modified version of the Morgan-Elson assay. Out of the 3950 bioactives, 9 were identified as lead compounds. All of the compounds identified from the bioactive collection are novel GFAT inhibitors.</p> <p>A structure-activity relationship (SAR) analysis was performed on the lead compounds. Derivatives of the leads were also purchased or synthesized for inhibitory testing. Four distinct classes of compounds were identified as GFAT inhibitors: isoquinolines, aminothiazoles, pyridinones and quinones. The most potent lead compound elucidated from the SAR was dehydroiso-β-lapachone (IC₅₀ 1.5±0.5 µM). The mode of inhibition of dehydroiso-β-lapachone was determined to be non-competitive for both binding domains of recombinant hGFAT1.</p> <p>To validate the lead compounds as inhibitors of native hGFAT1 and to determine their cell permeability, a cell based assay was developed. HepG2 cell cultures were treated with an inhibitor and HBP metabolism was determined by measuring the levels of the end-product uridine diphosphate <em>N</em>-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was separated and detected by UPLC-ESI-TOF-MS and metabolite levels were normalized to cell concentration. The leads, alloxan, lapachol and amrinone all displayed hGFAT1 inhibition in cell culture.</p> / Doctor of Philosophy (PhD)