Objective This study investigated the role of plasma adropin levels to show endothelial dysfunction in individuals with type 2 diabetes mellitus and to compare these with flow-mediated dilatation.
Methods A total of 92 individuals with diagnosed type 2 diabetes mellitus were included and divided into 2 groups according to their brachial flow-mediated dilatation. The endothelial dysfunction group consisted of 46 participants with flow-mediated dilatation change of less than 7%, whereas 46 participants with flow-mediated dilatation change of more than 7% were accepted as the nonendothelial dysfunction group. Venous blood samples were taken from all study participants, and plasma adropin levels were measured using an enzyme-linked immunosorbent assay kit.
Results The mean flow-mediated dilatation values were 13.2% ± 4.9% in the nonendothelial dysfunction group, and 3.5% ± 3.4% in the endothelial dysfunction group. Mean hemoglobin A1c levels were significantly higher in the endothelial dysfunction group than in the nonendothelial dysfunction group (8.7% ± 1.9% vs 7.9% ± 1.6%, respectively; P < 0.038). Mean plasma adropin levels were 3.04 ± 0.79 ng/mL in the endothelial dysfunction group and 4.67 ± 1.43 ng/mL in the nonendothelial dysfunction group; P < 0.001. Plasma adropin levels showed no correlation with body mass index (r = −0.072, P = 0.497) but were positively correlated with flow-mediated dilatation values (r = 0.537, P < 0.001). In the linear regression analysis, adropin and hemoglobin A1c were independent risk factors for endothelial dysfunction in individuals with type 2 diabetes mellitus.
Conclusion Adropin is a new marker for use in demonstrating endothelial dysfunction.
Diabetes mellitus (DM) is a chronic condition that increases cardiovascular disease mortality, and its incidence is rising. The World Health Organization anticipates that by 2030, there will be approximately 360 million individuals with diabetes, worldwide,1and those with poor glycemic control could experience microvascular and macrovascular complications earlier than those who manage their disease better.2The endothelium produces some components of the extracellular matrix, and a variety of regulatory mediators, such as nitric oxide, prostanoids, endothelin, angiotensin II, von Willebrand factor, tissue-type plasminogen activator, plasminogen activator inhibitor-1, adhesion molecules, and cytokines.3These mediators allow the endothelium to regulate vascular tone and permeability, the balance between coagulation and fibrinolysis, the composition of the subendothelial matrix, the extravasation of leukocytes, and the proliferation of vascular smooth-muscle and renal mesangial cells.3Endothelial dysfunction (ED) has a key role in the pathogenesis of diabetic vasculopathies and has gained increasing attention in the study of diabetes-associated cardiovascular complications.4Endothelial dysfunction has emerged as being the crucial early step in the development of atherosclerosis and vascular complications in diabetes. The progression of vasculopathy is highly dependent on the degree of hyperglycemia,5and ED is an independent predictor of cardiovascular events in people with type 2 DM.6
Endothelial function can be measured using isolated blood vessels and by studying the capability of endothelium-dependent vasodilators, such as acetylcholine. It can also be assessed in microvessels, under pressurized conditions wherein either the effects of endothelium-dependent vasodilator-mediated or flow-mediated dilatations (FMD) can be examined.7
Adropin is a recently identified protein that seems to regulate endothelial function.8,9It has been shown that the mean maternal and cord serum adropin levels in women with gestational DM were markedly lower than those in a control group of pregnant women.10The authors therefore concluded that lower adropin levels may play a role in the underlying mechanisms of gestational DM.10
In the light of these data, we discuss the role of adropin on ED in type 2 DM. We also aimed to investigate the compliance of plasma adropin levels with FMD to show ED in patients with type 2 DM.
MATERIALS AND METHODS
This was a prospective observational study, conducted in 92 individuals with diagnosed type 2 DM. Diabetes mellitus was defined as a fasting blood glucose level of 127 mg/dL or greater or a clinical diagnosis of diabetes, with patients using oral antidiabetic agents or on insulin treatment. The exclusion criteria were any known heart disease, the presence of congestive heart failure, a history of coronary artery disease, and other comorbid situations, such as acute or chronic renal failure, a history of acute infection within the previous 7 days, acute or chronic hepatic failure, hematologic disorder, presence of any chronic inflammatory and autoimmune disease, and any known malignancy. The study participants were enrolled according to their FMD values: the ED group consisted of 46 patients with FMD values less than 7%, and 46 patients with FMD values greater than 7% were accepted as the non-ED group. Age, sex, body mass index (BMI), waist length, and duration of DM were recorded. This study was approved by the ethics committee and the institutional review board of the Faculty of Medicine, Tokat Gaziosmanpasa University, Turkey; and informed consent was obtained from each participant.
Venous blood samples were taken after a minimum 8-hour overnight fast and 20 minutes of supine rest, drawn into ethylenediaminetetraacetic acid tubes, promptly centrifuged at 4°C, and frozen at −80°C until adropin analyses were undertaken. Plasma adropin levels were measured using an enzyme-linked immunosorbent assay kit (Phoenix Pharmaceuticals, Belmont, CA) according to the manufacturer’s instructions. The catalog number was EK-032-35. The detection range of the kit was 0.01 to 100 ng/mL, the sensitivity was 0.3 ng/mL, and the linear range was 0.01 to 100 ng/mL. The intra-assay (within day) and interassay (between days) coefficients of variations were less than 10% and less than 15%, respectively. Glucose was evaluated in serum by the glucoseoxidase method (Konelab-60I). Triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol concentrations were measured by an automated chemistry analyzer (Roche Diagnostics, Indianapolis, IN) using commercially available kits. Hemoglobin, red distribution width, neutrophilia, lymphocyte, platelet, and mean platelet volume were measured from tripotassium ethylenediaminetetraacetic acid–based anticoagulated blood samples and assessed with a Sysmex K-1000 (Block, Scientific, Bohemia, NY) autoanalyzer within 30 minutes of sampling.
Flow-Mediated Dilatation Method
B mode and Doppler Ultrasonography were used for FMD. Ultrasonographic measurements were performed at the onset of the study by cardiology specialists using a commercially available machine (Vivid 3 ® GE Medical System, Horten, Norway) with a 7.5-MHz linear array transducer. All examinations were performed in a quiet and temperature-controlled room. A sphygmomanometer was placed on the forearm to create flow stimulation in the brachial artery and was inflated until the systolic pressure was more than 50 mm Hg, thus stopping the antegrade blood flow and creating ischemia. Vasodilation consequently occurred at the resistance arteries distal to where the flow was blocked. When the sphygmomanometer was deflated, a reactive hyperemia occurred in the brachial artery. The second measurements of brachial artery were performed continuously from 30 seconds before, to the 90 seconds after, cuff deflation: (FMD, %) = (reactive hyperemia diameter − baseline diameter) / baseline diameters × 100).
We report continuous data as mean and standard deviation, or median and interquartile range. Categorical data were compared using the χ2 test, and comparisons between the 2 groups were carried out using an independent samples t test. Correlation analyses were performed using the Pearson or Spearman coefficient of correlation. Multiple linear regression analysis was applied to identify whether adropin was independently associated with ED, and P < 0.05 was accepted as being statistically significant. The trial version of SPSS 16 software program (demo version) was used for statistical analysis.
The baseline characteristics of the study participants are summarized in Table 1. Mean FMD values were 13.2% ± 4.9% in the non-ED group and 3.5% ± 3.4% in the ED group, whereas mean hemoglobin A1c (HbA1c) levels were significantly higher in the ED group than in the non-ED group (8.7% ± 1.9% vs 7.9% ± 1.6%, respectively; P < 0.038). The variables of mean age, sex, serum creatinine, fasting glucose, lipid profile, C-reactive protein, waist length, BMI, duration of diabetes, history of smoking, and hypertension were similar between the groups (Table 1).
Figure 1 shows the difference in plasma adropin levels between the 2 groups. Mean plasma adropin levels were 3.04 ± 0.79 ng/mL in the ED group and 4.67 ± 1.43 ng/mL in the non-ED group; P < 0.001.
Plasma adropin levels had no significant correlation with fasting glucose, HbA1c, triglyceride, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol, and no correlation with BMI (r = −0.072, P = 0.497). However, they were significantly positively correlated with FMD values (r = 0.537, P < 0.001).
In the linear regression analysis, adropin and HbA1c were independent risk factors for endothelial dysfunction (Table 2).
In the present study, we showed that plasma adropin levels were significantly lower in those participants with ED, as evidenced by FMD. We divided the participants into 2 groups: those with FMD values less than %7 formed the ED group, whereas those with FMD values greater than 7% formed the non-ED group. This study is of great importance because it is the first to compare the effects of plasma adropin levels and FMD technique on ED. We found that plasma adropin levels show ED as FMD, strengthening the evidence suggesting that adropin plays a role in ED.
The primary etiology for mortality and morbidity in patients with diabetes (type 1 or type 2) is vascular disease.11Type 2 DM affects small and/or large vessels. Retinopathy, neuropathy, and nephropathy are the hallmarks of microvascular disease, whereas macroangiopathy in diabetes is manifested by accelerated atherosclerosis in coronary and brain arteries. Endothelial dysfunction plays an important role in vascular complications in people with diabetes,12and the role that FMD plays is as complicated in type 2 DM as it is in type 1. The effects of aging, hyperlipidemia, hypertension, and other factors increase the complexity of the problem. A major pathophysiological alteration in type 2 DM is insulin resistance. As a result, a great research effort has been focused on defining the possible contribution of insulin resistance to FMD, and the two may be associated by a number of mechanisms, including disturbances of subcellular signaling pathways common to both insulin action and nitric oxide production. The roles of oxidant stress, endothelin, the renin-angiotensin system, and the secretion of hormones and cytokines by adipose tissue are the other potential mechanisms.12
Hyperglycemia is the major causal factor in the development of FMD in patients with DM.12In accordance with this, in our study, the patients in the ED group had HbA1c levels than the non-ED group. Hemoglobin A1c was an independent predictor for ED.
Endothelial dysfunction basically involves either an increase or a decrease in any of the endothelial cell–related chemical messengers. Some examples of ED include an increased permeation of macromolecules,13,14increased or decreased production of vasoactive factors producing abnormal vasoconstriction/vasodilation,14–16and increased prothrombotic and/or procoagulant activity.17Although FMD occurs in many different disease processes, oxidative stress can be identified as a common denominator.18,19
From the clinical perspective, endothelial function has been estimated by measuring changes in blood flow, either invasively20or noninvasively.21Endothelium-dependent vasodilatation can be assessed in the coronary and peripheral circulations. Physiologically, endothelial function is defined in vivo in humans as an increase in blood flow or in the diameter of a vessel in response to agents that increase the concentration of nitric oxide. Blood flow and/or vessel diameter can be measured using a wide array of techniques, and endothelial function can be further evaluated by using a physiological measurement of blood flow, coupled with blood-level determination of selected compounds thought to reflect endothelial function. Such compounds include endothelin,22von-Willebrand factor,23thrombomodulin,24selectin,25adhesion molecules,26and tissue plasminogen activator, as well as its inhibitor, plasminogen activator inhibitor-1.27
In coronary circulation, noninvasive tests for the assessment of coronary endothelial function include positron emission tomography, Doppler echocardiography, and phase-contrast magnetic resonance imaging. The criterion standard test for the evaluation of coronary endothelial function requires invasive coronary angiography.12In peripheral circulation, brachial artery is used to assess FMD. This method evaluates the capacity of endothelial cells, which respond to pathological vasoconstrictive stimuli via enhanced production and bioavailability of nitric oxide and consequent vasodilatation. This evaluation is thought to be particularly important in individuals at risk of arteriosclerosis or other cardiovascular diseases. The assessment of brachial artery FMD from ultrasound imaging has been developed and widely used for evaluation of endothelial function because of its noninvasiveness and feasibility. Impaired brachial FMD is related to the prevalence and extent of coronary atherosclerosis and predicts cardiovascular events.27,28Endothelial dysfunction has previously been shown with FMD in patients with type 2 DM.29,30
Adropin is a new protein that exerts a protective role on endothelial cells and has been referred to as a novel regulator for these cells.8We previously showed lower adropin levels with nitric oxide levels in patients with syndrome X whose main pathophysiology is FMD.9Given the important results obtained in our study, we wonder about the use of adropin on FMD that detected with brachial FMD.
In the present study, we classified the participants into 2 groups according to their FMD values. One group had impaired endothelial function, whereas the other had normal endothelial function. Whereas Celik et al.9and Butler et al.31observed a negative correlation between adropin and BMI, Lian et al.32found a positive correlation. In the present study, we did not find a correlation between adropin levels and BMI values. All participants had type 2 DM, and most of such individuals have higher BMI values than healthy age-/sex-matched people. Therefore, we did not find any difference in the BMI values between the 2 groups. However, adropin levels were lower in the ED group, so we showed that they are independently affected by ED. The primary limitation of our study is that we did not measure oxidative stress markers, which could have provided important pathophysiological information. However, the present study showed that adropin is a new, useful, and effective marker for noninvasive evaluation of endothelial function.