Atherosclerosis 175 (2004) 253–259 Lack of direct effect of moderate hyperleptinemia to improve endothelial function in lean rat aorta: role of calorie restriction Michela Zanetti a,∗ , Rocco Barazzoni a , Marta Vadori b , Marco Stebel c , Gianni Biolo a , Gianfranco Guarnieri a a Department of Clinical, Morphological and Technological Sciences (DSCMT), Clinica Medica, Ospedale di Cattinara, Strada di Fiume 447, Trieste 34100, Italy b Department of Biomedical Sciences, Institute of Pharmacology, University of Trieste, Trieste, Italy c CSPA Animal Facility, University of Trieste, Trieste, Italy Received 16 January 2004; received in revised form 30 April 2004; accepted 4 May 2004 Available online 19 June 2004 Abstract Leptin stimulates vascular sympathetic activity and NO release. The resulting effect of hyperleptinemia on endothelial function is unknown. Reduction of food intake associated with hyperleptinemia may mediate some of the vascular effects of leptin. This study was designed to assess the relative effects of short-term moderate caloric restriction and hyperleptinemia on vascular function in lean rat. Endothelium-dependent and -independent vasorelaxation, nitric oxide synthase (eNOS) expression and nitrite production were measured in lean rats receiving leptin subcutaneous infusion (-26% caloric intake, -4% body weight versus control) or moderate caloric restriction (pair-feeding) for one week. Plasma leptin was increased (P ≤ 0.05) by ∼300% in the leptin-infused group, while it decreased (P ≤ 0.05) by ∼40% in calorie-restricted animals. Both leptin infusion and calorie restriction resulted in improved (P < 0.05) endothelium-dependent vasorelaxation to acetylcholine (EC 50 : -6.1 ± 0.2 and -6.2 ± 0.2 versus -5.4 ± 0.2 in control) and unaltered endothelium-independent vasodilation to DEA-NONOate. Furthermore, in aortas from leptin-infused and calorie-restricted rats, expression of eNOS and nitrite production were increased (P ≤ 0.05) to similar extent compared to control animals. These findings suggest that moderate short-term calorie restriction with adaptive hypoleptine- mia has independent beneficial effects on endothelial function in lean animals by enhancing eNOS expression and function. In addition, physiological hyperleptinemia does not independently contribute to improved vascular function above and beyond the effect of calorie restriction. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Leptin; Calorie restriction; Endothelium; Nitric oxide 1. Introduction Leptin, an adipocyte-derived hormone, is an important regulator of energy metabolism [1] with relevant vascu- lar effects. Leptin may increase blood pressure through enhanced sympathetic activity [2,3]. In addition, it also exerts direct depressor effects on vascular tone by stim- ulating endothelial production of nitric oxide (NO) in a dose-dependent manner [3–6]. Lembo et al. reported that ex vivo incubation of aortas from sympathectomized rats in the presence of leptin results in endothelial-dependent ∗ Corresponding author. Tel.: +39 040 3994416; fax: +39 040 3994593. E-mail address: zanetti@units.it (M. Zanetti). vasorelaxation [5], and this effect is mediated by stimula- tion of the AKT-endothelial NO synthase phosphorylation pathway [6]. Ob/ob leptin-deficient mice are markedly obese and exhibit impaired endothelial-dependent vasore- laxation, which is reversed by leptin administration [7]. In spite of its potentially important role in the regulation of NO production and endothelium-dependent vasorelaxation, no studies have addressed sustained endothelial effects of leptin in healthy individuals. Leptin administration reduces appetite, resulting in calorie restriction and weight loss [1]. Calorie restric- tion per se results in improvement of the dysfunctional endothelium-dependent vasorelaxation associated with obe- sity [8,9], in the presence of concomitant reduction of circulating leptin [1]. In ob–ob leptin-deficient obese mice, 0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.05.001