Insulin and adiponectin inhibit the TNFα-induced ADMA accumulation in human endothelial cells: The role of DDAH
Introduction
There is growing evidence that endothelial dysfunction, reflected by an impaired nitric oxide (NO)-mediated vasodilatation [1], is an early step in the development of atherosclerosis [2]. The endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) is associated with reduced NO production and impaired endothelium-dependent vasodilatation [3]. Plasma ADMA concentrations have been found significantly increased in a variety of disease entities, including patients with chronic renal diseases and hypercholesterolemia [3], [4], [5], [6], [7]. Previous studies have demonstrated that ADMA concentrations are strongly associated with components related to the metabolic syndrome [7], [8], in which the clustering of factors such as obesity, dyslipidaemia, hypertension and insulin resistance contributes to a substantial increase in the risk of cardiovascular diseases [9], [10].
Hyperinsulinemia is an important component of the metabolic syndrome, and there is evidence that insulin per se has important direct effects on the vasculature, exerting both vasodilating and vasoconstricting effects [11], [12]. However, the exact mechanisms by which insulin exerts its effect on the vasculature are still not fully understood, and whether hyperinsulinemia contributes to endothelial dysfunction in insulin resistance remains debatable.
Adiponectin is secreted by adipocytes, and mimics several metabolic effects of insulin. Plasma concentrations of adiponectin are reduced in obese humans, and decreased concentrations are associated with insulin resistance and hyperinsulinemia [13]. In addition, adiponectin seems to have antiatherogenic properties, and decreased concentrations have been demonstrated in patients with coronary artery disease [14]. However, the direct relationship between adiponectin and endothelial dysfunction has not yet been fully explored.
Whereas glucose has been found to be stimulatory to ADMA accumulation in human endothelial cells [15], no such data exist with regard to insulin. The present study was therefore designed to investigate whether the formation of ADMA in cultured human endothelial cells is directly influenced by insulin or adiponectin in the ranges of human physiological concentrations. As plasma concentrations of insulin has been strongly correlated to ADMA concentrations in chronic insulin resistance states [8], [16], but was shown to reduce ADMA concentrations during acute hyperinsulinemia [17], we hypothesised that insulin might exert either stimulating or suppressing effects on ADMA accumulation in cell cultures. Based on the strong association previously shown between ADMA and obesity, another hypothesis was that adiponectin would decrease the formation of ADMA. Our final hypothesis was that any effects of insulin and adiponectin on ADMA concentrations might be regulated by the activity of the degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH).
Section snippets
Cell subculture preparation
Human umbilical vein endothelial cells (HUVECs), obtained from a caucasian female donor, and human coronary artery endothelial cells (HCAECs), obtained from a black male donor (Cambrex, Kerviers, Belgium, for both) were cultured according to the manufacturer's instructions and aliquoted in batches for use in the respective experiments. Briefly, the endothelial cells were seeded at a density of 5000 cells/cm2 and grown in endothelial basal medium (Cambrex) supplemented with human recombinant
Cell viability
Exposure of HCAECs and HUVECs to any of the stimulators at any concentrations did not influence cell viability (data not shown).
ADMA elaboration by endothelial cells
In both HCAECs and HUVECs, the concentration of ADMA in the conditioned medium of control cells increased with time from 3.0 ± 0.05 to 4.2 ± 0.1 (p < 0.01) and from 1.9 ± 0.04 to 3.1 ± 0.09 pmol/μg protein (p < 0.001), respectively, after 48 h. In response to TNFα, insulin and glucose significantly increased accumulation of ADMA in the conditioned medium was observed over time in
Discussion
In the present study our main findings were that in human cultured endothelial cells the TNFα-induced ADMA accumulation was markedly inhibited in the presence of insulin and adiponectin, both in a dose-dependent manner. These observations could be explained by changes in the endothelial DDAH activity. In accordance with the literature, our cell lines exposed to TNFα showed significant ADMA accumulation over time [18]. TNFα has a number of functions and reduced NO bioavailability in cultured
Acknowledgements
We want to thank Baard Birkenes and Theresa Mai for laboratory assistance.
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