Leptin-induced endothelial dysfunction in obesity

Abstract

Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) nanosensors were placed near the surface (5+/-2 microm) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, O(2)(-), ONOO(-) were recorded. Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, O(2)(-), or ONOO(-) release from HUVEC. However, a 12-h exposure of HUVEC to leptin increased eNOS expression and CaI-stimulated NO (625+/-30 vs. 500+/-24 nmol/l control) and dramatically increased cytotoxic O(2)(-) and ONOO(-) levels. The [NO]-to-[ONOO(-)] ratio ([NO]/[ONOO(-)]) decreased from 2.0+/-0.1 in normal to 1.30+/-0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration coincided with 100% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and a reduced l-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250+/-10 vs. 420+/-12 nmol/l control), and an elevated concentration of O(2)(-) (240%) and ONOO(-) (70%). L-Arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[ONOO(-)] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular l-arginine, resulting in eNOS an uncoupling and depletion of endothelial NO and an increase of cytotoxic ONOO(-). Hyperleptinemia triggers an endothelial NO/ONOO(-) imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and diabetes.

Fig. 1.

Typical amperometric curves showing nitric oxide (NO; A), superoxide (O₂⁻; B), and peroxynitrite (ONOO⁻; C) release from a single human umbilical vein endothelial cell (HUVEC). Leptin (0.1 μg/ml; a) or calcium ionophore (CaI; 1 μmol/l) was used to stimulate NO, O₂⁻, and ONOO⁻ generation from a HUVEC incubated for 2 h without leptin (b) or with leptin (0.1 μg/ml; c).

Fig. 2.

The effect of leptin on CaI (1 μmol/l)-stimulated peak concentration of NO (A), O₂⁻ (B), and ONOO⁻ (C) released from HUVEC. Cells were incubated with various concentrations of leptin for 2 h (▪) and 12 h (•). *P < 0.05 vs. control (without leptin); n = 5 experiments.

Fig. 3.

CaI (1 μmol/l)-stimulated peak concentration of NO (A), O₂⁻ (B), and ONOO⁻ (C) as a function of incubation time. HUVECs were incubated with leptin (0.1 μg/ml) (▪) or without leptin (•). *P < 0.05 vs. control (without leptin); n = 4 to 5 experiments.

Fig. 4.

A: maximal NO, O₂⁻, and ONOO⁻ concentration produced by HUVEC in the presence of O₂⁻ or ONOO⁻ scavengers. NO, O₂⁻, and ONOO⁻ releases were stimulated by CaI (1 μmol/l) from a HUVEC incubated for 12 h with leptin (0.1 μmol/l) or with polyethylene glycol (PEG)-SOD (100 U/ml) (gray bar) or MnTMPyP (0.02 mmol/l) (black bar); control (white bar), *P < 0.0001 vs. control; n = 4 to 5 experiments. B: the ratio (R) of peak NO concentration to the peak concentration of ONOO⁻ (R = [NO]/[ONOO⁻]). NO and ONOO⁻ releases were stimulated by CaI (1 μmol/l) from HUVEC incubated for 2 h without leptin (control, white bar) or incubated with leptin (0.1 μg/ml) for 2 h (gray bar) or 12 h (black bar) at normal and elevated l-arginine (l-Arg; 3 mmol/l) or l-arginine (3 mmol/l) and sepiapterin (0.1 mmol/l). *P < 0.05 vs. control and **P < 0.01 vs. 12-h incubation without l-arginine, sepiapterin; n = 4 to 5 experiments. C: CaI-stimulated maximal NO and ONOO⁻ concentration released from HUVEC in the absence (control, white bars) or in the presence (black bars) of N^G-nitro-l-arginine methyl ester (l-NAME; 0.3 mmol/l) and apocynin (3 mmol/l) or S-17834 (0.07 mmol/l). The concentration of NO and ONOO⁻ was measured after 12 h incubation with leptin (0.1 μg/ml). *P < 0.001 vs. control, n = 4 to 5 experiments.

Fig. 5.

A: endothelial NO synthase (eNOS) expression in HUVEC incubated with various concentrations of leptin for 2 h (white bars) and 12 h (black bars). *P < 0.05 vs. control (without leptin); n = 4 to 5 experiments. B: time-dependent effect of constant leptin concentration (0.1 μg/ml) on l-arginine level in HUVEC. Cells were incubated with leptin (▪) or without leptin (•) leptin for 12 h. *P < 0.05, n = 4 to 5 experiments.

Fig. 6.

A: body weight of C57BL/6J mice maintained on low-calorie diet (▪) or high-calorie (60 kcal fat) diet with (○) or without (•) supplementation of l-arginine. B: leptin concentration changes in blood plasma of C57BL/6J mice maintained on low-calorie diet (▪) or high-calorie D-12492 (60 kcal fat) diet with (○) or without supplementation (•) of l-arginine (•). *P < 0.05 vs. control mice (low-calorie diet); n = 4 to 5 mice. C: leptin vs. body weight linear plot (r = 0.93, P < 0.05) for the low-calorie diet (▪, solid line) or exponential plot (r² = 0.95, P < 0.0001) for high-calorie diet D-12492 (60 kcal fat) diet (○, dashed line).

Fig. 7.

A: eNOS expression in aortic endothelial cells of C57BL/6J mice maintained on a low-calorie diet (control, white bars) and high-calorie diet (60 kcal fat; black bars) for 105 Days. B: cGMP content in aortic wall of C57BL/6J mice maintained on a low-calorie diet (control, black bars) and a high-calorie diet (60 kcal fat; white bars) *P < 0.005, n = 4 to 5 mice. C: CaI (1 μmol/l)-stimulated NO, O₂⁻, and ONOO⁻ release from aortic endothelial cells of C57BL/6J mice maintained for 105 days on low-calorie diet (black bars) or high-calorie (60 kcal fat, white bars) with or without supplementation with l-arginine (100 mg·kg⁻¹·day⁻¹) or l-arginine (100 mg·kg⁻¹·day⁻¹) and sepiapterin (10 mg·kg⁻¹·day⁻¹). *P < 0.01 vs. control (low-calorie diet); #P < 0.05 vs. obese mice without l-arginine supplementation; n = 4 to 5 mice.

Fig. 8.

A: the ratio of NO concentration to the concentration of ONOO⁻ (R = [NO]/[ONOO⁻]). NO and ONOO⁻ releases were stimulated by CaI (1 μmol/l) from endothelial cells of the aortas of C57BL/6J mice maintained for 105 days on low-calorie diet (1) or on high-calorie (60 kcal fat) diet (2) with supplementation of l-arginine (100 mg·kg⁻¹·day⁻¹) (3) or with supplementation with l-arginine (100 mg·kg⁻¹·day⁻¹) and sepiapterin (10 mg/kg/day) (4). *P < 0.01 vs. control (low-calorie diet); #P < 0.05 vs. obese mice without supplementation with l-arginine or l-arginine and sepiapterin; n = 5 mice. B: l-arginine concentration in serum and aortic endothelium of C57BL/6J mice maintained for 105 days on low-calorie diet (1) or on high-calorie (60 kcal fat) without (2) or with supplementation with l-arginine (100 mg·kg⁻¹·day⁻¹) (3). *P < 0.01 vs. control (low-calorie diet); #P < 0.05 vs. obese mice without supplementation with l-arginine; n = 5 mice.