Leptin Infusion and Endothelial Vasomotor Response

Adipose tissue is an active endocrine organ producing several hormones with circulatory and metabolic effects. In 1994, the hormone leptin was discovered. The lack of this hormone explained extreme obesity in rare patients and parenteral substitution restored body weight and metabolic disturbances. It was however soon discovered that most humans had too high levels which were related to development of cardiovascular diseases and diabetes. It was hypothesised that leptin induced vessel dysfunction which could explain this association.

In this study, we wanted to examine the association between leptin and vessel function by using the venous occlusion plethysmography method. We used three protocols to evaluate this association.

First protocol. In ten healthy males, leptin was infused locally in the forearm and forearm blood flow (FBF) was measured.

Second protocol. In ten healthy males, leptin or normal saline was infused locally in the forearm and FBF was measured. Concomitantly, four vasodilatators were infused locally in the forearm in a randomised order and the response (blood flow and fibrinolysis) was measured.

Third protocol. In eighty-three patients with known coronary artery disease, three vasodilators were infused locally in the forearm in a random order and response (FBF and fibrinolysis) was measured. The response was related to endogenous leptin levels.

The two first protocols were performed in Umeå, Sweden whereas the third was performed in Edinburgh, UK, all in 2006.

Introduction High BMI and particularly fat mass index are associated with increased risk of coronary artery disease and other cardiovascular conditions, but the underlying mechanisms are not well understood. Endothelial dysfunction precedes atherosclerosis and represents an important link between obesity and cardiovascular events.

The adipose tissue produces cytokines and hormones (adipokines), which, in excess, may promote cardiovascular disease by proinflammatory, prothrombotic, dyslipidemic and atherosclerotic effects.

Leptin is an adipokine with pleiotropic effects and circulating leptin levels are positively associated with the amount of body fat. High plasma leptin levels (hyperleptinemia) associate with the development of atherosclerosis, hypertension and coronary artery disease (CAD). Leptin activates specific leptin receptors expressed, among other tissues, in vascular cells, suggesting that leptin may participate in the development of endothelial dysfunction and atherosclerosis.

However, the net effect of leptin on vasomotor function remains unclear, as both vasodilation and vasoconstriction have been reported. Leptin induces release of nitric oxide (NO) in vitro and elicits endothelium-dependent vasodilation in mice by inducing endothelial expression of NO synthase. In addition, studies in humans have shown that leptin infusion exerts vasodilatation. In contrast, others have shown leptin-induced vasoconstriction in vitro and impaired vasodilatation in dogs. Different mechanisms have been proposed causing increased peripheral vascular resistance, such as vascular inflammation, increased sympathetic nervous system (SNS) activity, increased endothelin-1 (ET-1) production, and decreased nitric oxide (NO) bioavailability.

Hyperleptinemia has been associated to states of altered fibrinolysis, which is common in diabetes, cardiovascular disease and obesity. However, whether leptin directly influences the endogenous fibrinolytic function remains unclear.

The aim of these studies was to evaluate the role of leptin on endothelial function in humans. For this purpose, the vasomotor and the fibrinolytic functions were assessed in healthy men during a state of pharmacologically induced hyperleptinemia. In a parallel study, the endothelial function was assessed in patients with established CAD and related to plasma leptin levels.

Material and Methods

Subjects Twenty healthy non-smoking male volunteers not taking any regular medication were recruited in Umeå, Sweden. 2). Eighty-three patients with established CAD were recruited from the cardiology outpatient clinic at the Royal Infirmary, Edinburgh, Scotland, and the characteristics of this cohort have been reported previously. These patients had stable angina and had a prior angiographic documentation of ≥50% luminal stenosis of at least one major epicardial coronary vessel. Written informed consent was obtained from each subject and the studies were carried out in accordance with the Declaration of Helsinki.

Venous occlusion plethysmography Subjects abstained from alcohol for 24 hours and from food, tobacco and caffeine-containing drinks for at least 4 hours before each study visit. All studies were carried out in a quiet temperature-controlled room maintained at 22-25 degrees Celsius (ºC). A 17-G venous cannula was inserted into the antecubital vein of each arm and the brachial artery of the non-dominant arm was cannulated with a 27-G needle (Cooper's Needle Works Ltd, UK). Bilateral forearm blood flow (FBF) was measured by venous occlusion plethysmography using mercury-in-silastic strain gauges. Blood pressure and heart rate were measured using a semi-automated non-invasive sphygmomanometer. To avoid acute vasomotor effects, all medications were withheld on the morning of each study.

Study design

Protocol 1 In ten healthy male volunteers, recombinant human leptin (Sigma-Aldrich, Saint-Louis, Missouri, USA) was infused intra-arterially at ascending doses of 80, 800 and 8,000 ng/min (6 minutes each). Heart rate, blood pressure, FBF, leptin, tissue plasminogen activator (tPA) antigen and plasminogen activator inhibitor type 1(PAI-1) antigen concentrations were determined at the end of each dose.

Protocol 2 In a double-blind randomized crossover study, ten healthy male volunteers received intra-arterial infusions of either leptin (800 ng/min) or saline on two separate occasions with at least 2 weeks between visits. FBF was measured in the infused and non-infused arms at baseline and at regular intervals during the one-hour leptin/saline infusion. Thereafter four vasodilators were infused concomitantly with intra-arterial leptin/saline infusions; bradykinin (endothelium-dependent vasodilator that releases tPA) at 100, 300 and 1,000 pmol/min (Clinalfa Ltd, Switzerland), acetylcholine (endothelium-dependent vasodilator that does not release tPA) at 5, 10 and 20 µg/min (Clinalfa Ltd, Switzerland), sodium nitroprusside (endothelium-independent vasodilator) at 2, 4 and 8 µg/min (David Bull laboratories, UK) and verapamil (endothelium-independent vasodilator) at 10, 30, 100 µg/min (Abbott UK Ltd) for 6 minutes at each concentration. Vasodilators were infused in a randomized order with a 15-minute saline washout period between each drug. Verapamil was always administered at the end because of its long-lasting vasomotor effects.

Venous blood was obtained from the infused and non-infused arms at baseline, before and during infusion of bradykinin, at 60 minutes and at the end of the study protocol.

Protocol 3 In patients with CAD (n=83), bilateral FBF was measured before and during intra-arterial infusions of substance P (endothelium-dependent vasodilator that releases tPA) at 2, 4 and 8 pmol/min (Clinalfa Ltd, Switzerland), acetylcholine at 5, 10 and 20 µg/min (as above) and sodium nitroprusside at 2, 4 and 8 µg/min (as above) for 6 minutes at each concentration. Bradykinin was not administered because many subjects were being treated with angiotensin-converting enzyme inhibition and this markedly potentiates its vasodilator and fibrinolytic effects. The vasodilators were administered in a randomized order with a 15-minute saline washout period between each drug. Venous blood samples were obtained before and during intra-arterial infusion of substance P to measure fibrinolytic markers.

Venous Sampling and Assays Fasting venous blood samples were drawn into tubes containing acidified buffered citrate or trisodium citrate. Samples were collected immediately onto ice and centrifuged at 2,000 g for 30 min. Platelet-free plasma and serum were stored at -80°C before assay. Brain natriuretic peptide (BNP), cholesterol and glucose concentrations were determined according to clinical routine, and high sensitivity C-reactive protein (hsCRP) with a highly sensitive assay using particle-enhanced immunonephelometry (Behring BN II nephelometer). Plasma leptin concentrations were measured using a double-antibody radioimmunoassay (Millipore, Billerica, Massachusetts, USA). According to the literature, intra- and inter-assay coefficients of variation should be less than 5% at both low (2-4 ng/mL) and high (10-15 ng/mL) leptin concentrations. Plasma tPA and PAI-1 antigen concentrations were determined using enzyme-linked immunosorbent assays (Coaliza®, Chromogenix Ltd) and plasma tPA activity using a photometric method (Coatest tPA, Chromogenix Ltd). According to the manufacture, the coefficients of variation for fibrinolytic assays are 5.9% and 12% for tPA antigen and activity respectively, and 6.2% for PAI-1 antigen. Estimated net release of tPA (antigen and activity) was calculated as previously described after each dose of bradykinin or substance P, as the product of the infused forearm plasma flow and the difference in plasma levels between the infused and non-infused forearms.

Eligibility Criteria:
Inclusion criteria protocol 1 and 2;

* Healthy male
* No regular medication
* Non-smoking
* Abstain from alcohol for 24 hours and from food, tobacco and caffeine-containing drinks for at least 4 hours before each study visit

Inclusion criteria protocol 3;

* Established coronary artery disease
* Stable angina pectoris
* Documented ≥ 50% stenosis of at least one major epicardial coronary vessel

Exclusion criteria protocol 3;

* Coronary revascularisation within three months
* Diabetes mellitus
* Cardiac failure (ejection fraction \<35% or New York Heart Association (NYHA) ≥2)
* Renal impairment (creatinine ≥200 µmol/L)
* Systolic blood pressure \<100 or \>190 mmHg