Introduction
Metabolic syndrome is a cluster of disorders of obesity, dyslipidemia, hypertension, insulin resistance, and proinflammatory and prothrombotic states. A major consequence of this syndrome is the increased risk of cardiovascular diseases which makes metabolic syndrome an epidemic public health problem [
1].
The key feature of metabolic syndrome is the systemic insulin resistance. In addition to its known effects on metabolisms, insulin also regulates cardiovascular functions, acting as a vasodilator primarily by increasing endothelial nitric-oxide synthase (eNOS) gene expression and activity and thus, nitric oxide (NO) bioavailability, which in turn engenders an array of anti-atherogenic actions [
2].
The pathogenesis of insulin resistance is so complicated that several factors, such as obesity, a proinflammatory state, aging, and genetic variants, contribute to its development. Recently it has become clear that inflammatory factors can inhibit the IRS/PI3K/Akt pathway and lead to insulin resistance [
3]. However, molecular mechanisms connecting inflammation and insulin resistance are not definite. Resistin, a 12.5-kDa cysteine-rich peptide protein, is originally described as an adipocyte-derived polypeptide which can cause insulin resistance in rodents [
4]. Increasing evidence however suggested that resistin may be involved in the inflammatory process, which is a common feature in metabolic syndrome, insulin resistance status, and vascular diseases. Resistin has a structural similarity to many proteins involved in the inflammatory processes, especially to a family of proteins named “found in inflammatory zone” (FIZZ) or “found in resistin-like molecule” (RELM) [
5]. Consistent with the hypothesis that resistin plays a role in the inflammatory process, recent studies have shown that resistin is abundantly expressed in mononuclear leukocytes, macrophages, spleen, and bone marrow cells in human subjects [
6]. Resistin expression can be increased by proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor-α and C-reactive protein) [
7]. Indeed, resistin can also up-regulate interleukin-6 and tumor necrosis factor-α and induce arthritis [
8].
In this study, we investigated the hypothesis that resistin, as an inflammatory mediator, may impair insulin signaling in the endothelium, which leads to eNOS activity inhibition and endothelial dysfunction.
Materials and methods
Materials
Human recombinant resistin was obtained from Phoenix. Inc, USA. Endothelial cell growth factors (ECGs) were purchased from Santa Cruz, USA. Medium 199, pancratine and anti-human von Willebrand factor IgG were from Gibco, USA. Fetal bovine serum(FBS) was procured from Hyclone (Logan, Utah, USA). Phospho-Akt (Ser473), Anti-rabbit-IgG-HRP and phospho-eNOS (Ser1177) were from CST (Beverly MA, USA). 4, 5-Diaminofluorescein Diacetate (DAF-2DA) was obtained from Cayman (Ann Arbor, Michigan, USA).
Endothelial cell culture
Human umbilical endothelial cells (HUVECs) were harvested enzymatically from the umbilical cords of healthy parturients under sterile conditions, as described by Minter
et al [
9]. Endothelial cell monolayers were grown to confluence in M199 containing 20% FBS, 75 μg/mL endothelial cell growth supplement (ECGS), 0.1 mg/mL heparin, 50 U/mL penicillin, and 50 μg/mL streptomycin at 37°C in 5% CO
2. Cells were passaged every 2-3 days and cells from passages 3 to 5 were used for experiments. Cells were identified as endothelial cells by their typical cobblestone morphology and by immunostaining for the von-Willebrand factor.
Treatment
Cells from passages 3 to 5 were exposed to resistin (15, 50, 100 ng/mL) for 24 h. For insulin stimulation, confluent cells were exposed to resistin for 24 h and then starved in 0.5 % fetal calf serum (FCS), endothelial basal medium (EBM) with 15, 50, 100 ng/mL resistin for 6 h before stimulating with 100 nmol/L insulin for 20 min.
Western blot analysis
Treated HUVECs were collected, washed with ice-cold phosphate buffered saline, and lysed in protein lysis buffer [20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), 1 mmol/L ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerol phosphate, 1 mmol/L Na3VO4, 10 µg/mL each protease inhibitor (aprotinin, leupeptin, and pepstatin), and 1 mmol/L phenylmethylsulfonyl fluoride] for 1 h on ice. Protein concentration was measured by the Bradford method (Bio-Rad). Fifteen µg of protein per lane was separated by 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked by 5% nonfat powdered milk in Tris buffered saline with Tween 20 (TBST) (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.05% Tween 20). The membrane was incubated with the primary antibody in 2% powdered milk in TBST, washed extensively with TBST, and then incubated with the secondary anti-rabbit horseradish peroxidase-labeled antibody. Bands were visualized with enhanced chemiluminescence (ECL).
NO production measurement
To measure intracellular NO production, the cell-permeable fluorescent NO indicator 4, 5-diaminofluorescein diacetate (DAF-2DA) was used. This membrane-permeable dye is hydrolyzed intracellularly by cytosolic esterases releasing DAF-2, which is converted in the presence of NO into a fluorescent product, DAF-2 triazole. HUVECs were incubated in 5 µmol/L DAF-2DA solution for 30 min at 37°C in the dark and then washed. Cells were stimulated for 20 min in insulin (100 nmol/L) and then washed and fixed before examination under a confocal microscope (excitation wavelength, 485 nm; emission wavelength, 515-560 nm). Green fluorescence intensity was quantified.
Statistical analysis
All quantitative variables are presented as . The differences of three groups or more were analyzed by using SPSS 11.0, and P < 0.05 was considered statistically significant.
Results
The morphology of HUVECs
The morphology of attached cells observed by light microscope was a monolayer of flat, polygonal-shaped cells. Von-Willebrand factor immunostaining showed that there were many yellow-brown granulas in the cytoplasm.
NO production of HUVECs
Without insulin stimulation, NO production of HUVECs treated with 15, 50, 100 ng/mL resistin was similar to control cells (without resistin treatment), and there were no significant differences between any two groups. However, in insulin stimulated cells, NO production of HUVECs treated with 15, 50, 100 ng/mL resistin was significantly lower than control cells, and there was no difference in NO production between each concentration of HUVECs treated with resistin (Table 1).
Effect of resistin on Akt and eNOS phosphorylation
The Akt phosphorylation levels in HUVECs treated with 15, 50 and 100 ng/mL resistin but without insulin stimulation were similar to the control group (Fig. 1), and there were no significant differences among resistin groups with various concentrations. By insulin stimulation, the Akt phosphorylation levels in HUVECs treated with 15, 50 and 100 ng/mL resistin were significantly lower than the control group (P < 0.05) (Fig. 2).
Without insulin stimulation, eNOS phosphorylation levels in HUVECs treated with 15, 50 and 100 ng/mL resistin were significantly lower than that in the control group (P < 0.05) (Fig. 3), and so were the HUVECs treated with 15, 50, 100 ng/mL resistin and stimulated by insulin (P < 0.05) (Fig. 4) (Table 2).
Discussion
Increasing evidence suggests that inflammation causes insulin resistance, impairs endothelial function, and initiates or promotes pathogenesis of vascular diseases [
10,
11]. Resistin, as a potent inflammatory regulator, may affect insulin signaling and endothelial functions. Endothelial dysfunction and eNOS deregulation are the prominent features in metabolic syndrome and constitute the early events in atherogenesis. However, little data is available, about whether resistin could interfere with endothelial functions and insulin signaling in endothelial cells.
Our study showed that human recombinant resistin inhibits insulin-stimulated eNOS phosphorylation and NO production. Because NO is a key regulator in endothelial functions, eNOS inhibition by resistin could provide a mechanism for endothelial dysfunction, which is consistent with recent reports of the elevated resistin levels in patients with coronary artery disease [
12]. Moreover, Shen
et al [
13] reported that resistin inhibited insulin signaling and eNOS activation in human aortic endothelial cells. They found that eNOS phosphorylation at the Ser1177 was inhibited by 10-100 ng/mL resistin in a dose-dependent manner, indicating that resistin may restrain eNOS activity by inhibiting eNOS phosphorylation at Ser1177. Together with other findings that resistin can up-regulate the expression of adhesion molecules [
14,
15] and promote smooth muscle cell proliferation [
16], we speculated that resistin might provide a causal link for accelerated cardiovascular diseases in metabolic syndrome.
Although human and murine resistins share only 53% homology, a previous study showed that human resistin has similar functions as murine resistin [
17]. It has been reported that resistin forms a complex multimeric structure, which may be an obligatory step towards activation [
18]. Therefore, whether the recombinant resistin is physiologically relevant has been questioned. Firstly, human recombinant resistin used in this study has the dimer structure through disulfide bonds; secondly, our studies and that of others have shown that the recombinant resistin can exert pathological effects.
It is established that eNOS activity can be regulated by post-translational modifications through different signaling pathways, including the extracellular signal-regulated protein kinase, mitogen-activated protein kinase (ERK/MAPK) pathway [
19], AMP-activated protein kinase (AMPK) pathway [
20] and Akt pathway. Insulin can induce eNOS activation through the IRS/PI3K/Akt cascade, which plays a key role in insulin signaling in target tissues. Our results showed that resistin inhibited eNOS phosphorylation and NO production through restraining the Akt pathway in insulin stimulated HUVECs. However, without insulin stimulation, decreased eNOS phosphorylation was not accompanied by down-regulation of Akt phosphorylation, which was possibly explained by the mechanisms of other pathways of resistin. Resistin has been reported to impair insulin action in other target tissues [
21-
23]. However, reports on the mechanisms for inhibitory effects of insulin pathways are less consistent. Some studies demonstrated that insulin inhibited insulin-stimulated glucose uptake into muscle cells without affecting insulin signaling pathways [
24]. Some studies have even reported that resistin can induce Akt phosphorylation [
16,
25]. Several possibilities could explain this discrepancy, consisting of cell-specific effects and diversities in the experimental conditions in different studies. Indeed, our experiments have shown that resistin decreased Akt phosphorylation after 24 h of resistin treatment. Shen
et al [
13] reported that resistin induced a rapid stimulation of Akt phosphorylation to reach a peak in 30-60 min, but which returned to the basal level within 120 min and was followed by a decreased Akt phosphorylation, then reached the maximal effect in 48 h. So the inhibitory effect of resistin on Akt phosphorylation may reflect the true effects of chronic resistin elevation in the dynamic regulation of insulin signaling and Akt activity in metabolic syndrome.
In this study, we further demonstrated that resistin could inhibit NO productions in insulin stimulated HUVECs. However, NO production was not altered by resistin treatment in cells without insulin stimulation. Preliminary observations suggest that mean plasma resistin levels in patients with diabetes are about 40 ng/mL [
26]. And Burnett
et al [
27] reported that patients diagnosed with premature coronary artery disease (PCAD) were found to have higher serum levels of resistin than normal controls. These all indicate that elevated resistin plasma levels may inhibit insulin mediated endothelium NO production in diabetic patients.
In a word, our experiments showed resistin exerted an inhibitory effect on insulin signaling, eNOS activation and NO production in endothelial cells. Further, resistin-induced insulin resistance and eNOS inhibition in endothelial cells may play important roles in the pathophysiology of cardiovascular disease, such as hypertension and atherosclerotic process in metabolic syndrome. These findings may help to develop pharmacological agents to effectively prevent endothelial dysfunction associated with insulin resistance.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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