Is the HBP Indicated in the Inflammatory Response to TNFα?

BACKGROUND Inflammation enhances oxidative stress and plays a key role in the development of insulin resistance, diabetes (T2DM), endothelial dysfunction (ED), and hypertension (HTN). The hexosamine biosynthesis pathway (HBP) plays a vital role in cell health through protein activity, transcription, and cell signaling. One stimulus for upregulation of the HBP is excessive oxidative stress, disruption of glycolysis, and ultimately an increase in the pathway’s substrates. Addition of the HBP’s end‐product onto proteins, creating β‐linked N‐acetylglucosamine (O‐GlcNAc), is increased with oxidative stress, has been found to be higher in T2DM, and may specifically be important in the activation phase of the inflammation response. Additionally, increased O‐GlcNAcylation of endothelial nitric oxide synthase (eNOS) decreases the enzyme’s phosphorylation and production of the potent vasodilator nitric oxide (NO). Therefore, we investigated whether an inflammatory insult can increase the flux through this pathway and total O‐GlcNAcylation within endothelial cells (ECs). Because African Americans (AA) exhibit higher levels of ED and chronic inflammation than their Caucasian (CA) counterparts, potential racial differences were also examined. METHODS Human umbilical vein endothelial cells (HUVECs), n=6, from donors free of disease were either treated as a control or stimulated with 10 ng/mL of tumor necrosis factor alpha (TNFα) for 4 hours. Cell lysate was harvested, and immunoblotting procedures used to measure protein expression of the HBP rate‐limiting enzyme (GFAT), the transferase that adds the end‐product onto proteins (OGT), and total protein O‐GlcNAcylation (O‐GlcNAc). As a measure of EC health, total and activated eNOS (p‐eNOS) were measured. HUVECs from CA and AA donors were compared. RESULTS With TNFα stimulation, there was no difference in GFAT (0.63 ± .09 vs 0.62 ± .08, p=.89), OGT (0.49 ± .09 vs 0.41 ± .08, p =.56), nor total O‐GlcNAc (1.56 ± .38 vs 1.77 ± .42, p =.71) expression compared to controls. Neither eNOS, nor p‐eNOS were altered with inflammation compared to controls (1.25 ± .84 vs 1.28 ± .93, p =.90) and (.31 ± .15 vs .30 ± .15, p = .735), respectively. However, there were baseline racial differences in p‐eNOS expression that were retained with TNFα stimulation ( p <.01). No racial differences were found among any of the other proteins examined ( p >.05). CONCLUSION The HBP was not altered with acute inflammation in ECs, with no racial differences additionally found. Although this inflammatory stimulus did not alter eNOS, the enzyme responsible for endothelial NO production, ECs from AA expressed lower amounts of the activated enzyme basally and when stimulated. Low‐grade inflammation vitally contributes to ED, HTN, T2DM, and their complications. Because the HBP plays a role in each, it was examined as a possible contributor to the effects of inflammation. We did not find any link between the two and conclude that hyperglycemia may be necessary to work in conjunction with inflammation to increase protein O‐GlcNAcylation in ECs. Future research is needed to examine links between this pathway and additional conditions. Because we did not see a treatment effect with either of the proteins analyzed, a greater inflammatory stimulus may also be needed.Hexosamine Biosynthesis Pathway protein expression in response to inflammation. Densitometric quantification normalized to housekeeping protein. n= 4 independent experiments, 6 cell lines. Data shown as mean + SEM.Racial differences of indicators of vascular function in response to inflammation. Densitometric quantification normalized to housekeeping protein. n=4 independent experiments, 6 cell lines. Data shown as mean + SEM.