Palmitate evokes ceramide-dependent reactive oxygen species (ROS) generation from sources other than nadph oxidase in bovine aortic endothelial cells (BAECS)

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Title Palmitate evokes ceramide-dependent reactive oxygen species (ROS) generation from sources other than nadph oxidase in bovine aortic endothelial cells (BAECS)
Publication Type honors thesis
School or College College of Health
Department Exercise & Sport Science
Author Pettey, Dix Hale
Date 2010-05
Description In preliminary studies we showed that basal and insulin-mediated endothelial nitric oxide synthase phosphorylation (p-eNOS) and nitric oxide (NO) production are impaired when Bovine Aortic Endothelial Cells (BAECs) are treated with the saturated fatty acid palmitate (pal, 500 uM) vs. vehicle (veh) for 3 hours (h). Co-incubation of BAECs with the ceramide synthesis inhibitor myriocin (myr, 10 uM) negated these effects. Insulin-evoked 1.73 ± 0.09-fold increases in p-eNOS SI 177 that were abolished by pal, but were restored by concurrent treatment with myr (western immunoblotting, n=T6-32, p<0.05). Thus, pal incubation impairs insulin-stimulated p-eNOS SI 177. 100 nM insulin evoked 1.65 ± 0.10-fold increases in NO production by BAECs. These increases were negated by pal, and were restored by concurrent treatment of pal with myr (amperometric probes, n=9-31, p<0.05). Thus, we determined that palmitate incubation impairs insulin-stimulated NO production. ROS can lower NO bioavailability. The purpose of this study was to determine whether pal increases ROS production in a ceramide-dependent manner and if so, to determine the source of ROS production. DCFDA fluorescence was used to assess ROS after BAECs were incubated for 3h with veh ± pal, myr, 10 uM potassium cyanide (KCN, a mitochondrial ROS inhibitor), or 1 uM N-vanillylnonanamide (NVN, an NADPH oxidase inhibitor). Relative to veh, palevoked increases (2.8±0.1-fold, p<0.05, n=48) in ROS production were blunted (15±5%; p<0.05) but were not completely abolished by myr, suggesting the existence of ceramidedependent and -independent pathways. KCN, NVN, and KCN + NVN lowered (p<0.05) pal-evoked ROS production by 37±4%, 18±7%, and 39±12% (n=8-32), respectively, suggesting that in addition to mitochondrial and NADPH oxidase-dependent sources of ROS, additional sources of pal-induced ROS exist. Pal-induced ROS production decreased further when BAECs were co-incubated with myr + NVN (46±4%, p<0.05, n=16) vs. NVN alone, but not when myr was co-incubated with KCN (n=12) or KCN + NVN (n=16). Moreover, pal-evoked increases (p<0.05, n=12) in NADPH oxidase activity were similar in the absence (1.6±0.2-fold) and presence (2.1±0.2-fold) of myr. Thus, pal-evoked and ceramide-mediated ROS production in BAECs is not derived from NADPH oxidase but may arise from mitochondria.
Type Text
Publisher University of Utah
Subject Insulin resistance
Dissertation Institution University of Utah
Dissertation Name Honors BS
Language eng
Relation is Version of Digital reproduction of "Palmitate evokes ceramide-dependent reactive oxygen species (ROS) generation from sources other than nadph oxidase in bovine aortic endothelial cells (BAECS)" J. Willard Marriott Library Special Collections RC39.5 2010 .P48
Rights Management © Dix Hale Pettey
Format application/pdf
Format Medium application/pdf
Format Extent 221,931 bytes
Identifier us-etd2,184063
Source Original: University of Utah J. Willard Marriott Library Special Collections
Conversion Specifications Original scanned on Epson GT-30000 as 400 dpi to pdf using ABBYY FineReader 9.0 Professional Edition.
ARK ark:/87278/s61n8fn9
Setname ir_etd
ID 192904
Reference URL https://collections.lib.utah.edu/ark:/87278/s61n8fn9
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