Differences between acute and chronic leptin‐stimulated signaling and nitric oxide production in vascular endothelial cells

Acute leptin stimulation increases endothelial nitric oxide (NO) production in vitro. In contrast, in obese patients chronic leptin elevations are associated with endothelial dysfunction and impaired NO production. Therefore, the current study examined effects of chronic leptin stimulation on NO production in human aortic endothelial cells (HAEC). HAEC were treated with 5 or 60 ng/mL leptin for 0.5‐72 h. HAEC NO release was assessed by chemiluminescence. Superoxide (O2‾∙) production was measured with electron spin resonance spectroscopy. Endothelial NO synthase (eNOS) mRNA was determined using RT‐PCR. Protein levels of eNOS, p‐eNOS (ser1177), p‐Erk, suppressor of cytokine signaling (SOCS3), xanthine oxidase (XO) and selected NADPH oxidase subunits were examined by Western blots. Although acute leptin stimulation increased eNOS and Erk phosphorylation, chronic leptin stimulation was not cytotoxic to HAEC and failed to alter eNOS mRNA levels, eNOS phosphorylation, or NO release. Further, chronic leptin stimulation failed to alter O2‾∙ production, NADPH oxidase or XO expression, but did increase SOCS3 expression. These findings suggest that unlike acute stimulation, chronic leptin stimulation does not alter critical components of HAEC NO or O2‾∙ metabolism. We postulate that increased SOCS3 expression may attenuate vascular endothelial responses to chronic leptin stimulation