Effects of The Hexosamine Biosynthesis Pathway in Endothelial Function
Type of DegreePhD Dissertation
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Diabetes is characterized by high circulating plasma glucose and insulin resistance. Many diabetics have additional comorbidities, a major one being hypertension. Endothelial dysfunction links diabetes and hypertension. The Hexosamine Biosynthesis Pathway (HBP) is a route of excess glucose utilization that has been shown to decrease bioavailable nitric oxide, which is critical for proper vascular function and health. The end product of the HBP increases the addition of beta linked N-acetyl-glucosamine (O-GlcNAc) to various cytosolic, nucleic, and mitochondrial proteins. O-GlycNAcylated proteins compete with phosphorylation at many sites, contributing to the role of the pathway in cellular regulation. People with diabetes, chronic inflammation, and other pathological conditions see increased flux through the HBP. Because exercise has been shown to positively affect this pathway in vivo, an exercise memetic of shear stress was utilized to examine if the pathway is affected in the conditions above using a human umbilical vein endothelial cell (HUVEC) model. African Americans experience higher levels of diabetes, hypertension, and endothelial dysfunction in the population, therefore potential racial differences on pathway activation were examined. Researchers hypothesized that HUVECs from African American donors will show a greater flux through the HBP than those from Caucasian donors, and that shear stress will decrease the flux and negative effects of the pathway in cells from both African American and Caucasian donors. HUVECs were incubated for 24 hrs. with either high glucose (30mM) or normal glucose (5.5mM) as a control. Some cells were exposed to 24 hrs. of low or high physiological levels of shear stress to represent an athero-prone or athero-protective vascular profile, respectively. Western blotting techniques were used to measure protein expression of GFAT, OGT, and O-GlcNAc protein, as well as additional markers of endothelial function/ dysfunction. Markers of the pathway were not altered in hyperglycemic conditions or with varying levels of shear stress within HUVECs. There was no interaction of race and condition, but main effects of race were found in protein expression of vascular health markers VCAM and Caspase-3.