Attenuation of signaling and nitric oxide production following prolonged leptin exposure in human aortic endothelial cells.
Acute leptin exposure stimulates endothelial nitric oxide (NO) production in vitro. In contrast, chronic elevations in circulating leptin levels in patients with obesity are associated with endothelial dysfunction and impaired endothelial NO production. Therefore, the goal of the current study was to examine the direct effects of acute and more sustained leptin stimulation on endothelial nitric oxide synthase (eNOS) and NO production in human aortic endothelial cells (HAECs). HAECs were treated with vehicle or with leptin (5 or 60 ng/mL) acutely (30-60 minutes) or for 72 hours. HAEC NO release into culture media was measured with a chemiluminescence technique, and superoxide (O(2)(-.)) production was measured with electron spin resonance (ESR) spectroscopy. HAEC eNOS activity was measured as the conversion of (3)H-arginine to (3)H-citrulline, and protein levels of eNOS, phospho-eNOS (serine 1177), Erk, phospho-Erk, suppressor of cytokine signaling (SOCS3), xanthine oxidase (XO), and the reduced nicotinamide adenine dinucleotide (NADPH) oxidase components p22phox, p67phox, Nox-4, and gp91phox were examined by Western blotting or immunoprecipitation. Acute leptin exposure increased eNOS serine 1177 phosphorylation and caused Erk activation. In contrast, prolonged leptin stimulation was not cytotoxic and failed to alter eNOS expression, phosphorylation, or HAEC NO release. Furthermore, prolonged leptin stimulation did not alter O(2)(-.) production or NADPH oxidase or XO expression but increased SOCS3 expression. In contrast to acute stimulation, prolonged (72 hours) stimulation does not alter endothelial cell NO or O(2)(-.) production. We postulate that chronic leptin stimulation, through increased SOCS3 expression, may attenuate the effects of leptin on vascular endothelial function.
Department of Medicine, Emory University and Atlanta Veterans Affairs Medical Centers, Atlanta, GA 30033, USA.
Carmelo
C
Blanquicett
Anitra
A
Graves
Dean J
DJ
Kleinhenz
C Michael
CM
Hart
D001011:Aorta / Q000166:cytology / Q000187:drug effects*; D018929:Cell Culture Techniques; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D020738:Leptin / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*
D020738:Leptin; D009569:Nitric Oxide