Does flux through the hexosamine biosynthesis pathway differ between endothelial cells from African American and Caucasian donors?

Abstract

People with Type 2 Diabetes (T2D) often exhibit endothelial dysfunction (EndoDys), which underlies the initiation of many diseases. African Americans (AA) have a greater prevalence of T2D and EndoDys than their Caucasian (CA) counterparts. To understand these differences, we examined glucose metabolism within the endothelium. Specifically, we sought to determine whether there are differences between AAs and CAs in the Hexosamine Biosynthesis Pathway (HBP), a route of glucose utilization that is upregulated in T2D. We hypothesized that endothelial cells (EC) from AA donors will show a greater flux through the HBP and express higher markers of EndoDys compared to CA donors. Human umbilical vein ECs (HUVECs), n=2-3/group, were incubated for 24hrs with high (HG, 30mM) or normal (5.5mM) glucose. Protein expression was measured via western blot for the: 1) HBP rate-limiting enzyme (Glutamine: fructose-6-phosphate amidotransferase, GFAT); 2) enzyme catalyzing the addition of HBP end-products to various proteins (O-GlcNAc transferase, OGT); and 3) attachment of O-linked N-acetylglucosamine moieties to various proteins (O-GlcNAcylation, O-GlcNAc), in addition to markers of EndoDys. Hyperglycemia did not differentially impact the expression of HBP markers GFAT or OGT between ECs from CA and AA donors (glycemic status [GS], p=0.6723; race, p=0.3807 and GS, p=0.8695; race, p=0.3536, respectively). Although total O-GlcNAc did not significantly increase with HG, there was a trend toward higher O-GlcNAcylation in AA ECs (p=0.0897). Upon further examination of O-GlcNAcylation, there were 3 prominent bands of highly O-GlcNAcylated proteins at >220kDa (band 1), ~150-200kDa (band 2), and ~85kDa (band 3). There was significantly higher O-GlcNAcylation of protein in band 2 for AA donors compared to CA donors (p=0.0362). The apoptotic marker, Caspase-3, did not change with HG (p=0.9219) nor differ in expression between races (p=0.2060). The inflammatory marker, VCAM, was significantly higher in cells from AA donors (p=0.002) but did not differ with GS (p=0.3468). There was no GS by race interaction found. EndoDys links T2D and other pathologies. We examined the HBP pathway of glucose utilization in ECs from CA and AA donors. There was a main effect of race related to inflammation and total O-GlcNAcylation, without differences in the expression of key proteins within the pathway. Our results suggest that the HBP may contribute to the racial differences in EndoDys, however, this relationship is not straightforward and does not seem to be exacerbated in the context of HG. Future research is needed to examine these unexpected discrepancies and interrogate the link between HBP flux within the endothelium, along with its connection to various pathologies This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.