Table of Contents
Ethical approval
Ethical approval was obtained from the Clinical Research Ethics Committee of Istanbul University, Cerrahpaşa Medical Faculty (Approval Date-Number: 21/06/2016-227647). Written informed consent was obtained from all participants before their inclusion in the study, ensuring that they were fully informed about the study’s purpose, procedures, and their rights.
Subjects
This observational, cross-sectional study was conducted between July 2016 and October 2017. Patients diagnosed with T2DM were included in the study according to the diagnostic criteria established by the American Diabetes Association (ADA).
Classification of vascular complications
T2DM patients were grouped based on the predominant type of vascular complication. Microvascular complications included diabetic retinopathy (ETDRS-based diagnosis), neuropathy (confirmed by ENMG), and nephropathy (based on albuminuria and/or reduced eGFR). Macrovascular complications included CAD, peripheral artery disease, or cerebrovascular events, confirmed by clinical and imaging findings. In patients with both types, classification was based on the dominant clinical presentation. This stratification aimed to explore whether adropin and miR-21 levels differ according to the primary vascular involvement.
We divided the study cohort into five distinct groups1: Diabetic Group [DM] (n= 45), including patients without complications, mean age: 52.9 ± 9.5 years; females: 30, males: 152; Diabetic Group with Microvascular Complications (n= 24) with mean age: 51.8 ± 6.4 years; females: 17, males: 73; Diabetic Group with Macro-vascular Complications (n= 20) with mean age: 55.7 ± 3.4 years; females: 8, males: 124; Coronary Artery Disease without Diabetes Mellitus (n = 19) with mean age: 51.8 ± 7.4 years; females: 11, males: 8; and5 Healthy Control Group [HC] (n = 20), including persons without any diagnosed endocrine, cardiovascular, and inflammatory disorders with mean age: 47.8 ± 4.7 years; females: 9, males: 11. As this was an observational study, no randomization or blinding was applied (Fig. 1).
Flow diagram of participant selection and grouping.
In the study, patients with T2DM were categorized into three subgroups. The uncomplicated diabetes group included patients without any diabetes-related complications at the time of inclusion. The microvascular diabetes group comprised patients with at least one microvascular complication, while the macrovascular diabetes group consisted of patients with at least one macrovascular complication. Individuals were excluded if they had acute or chronic metabolic, systemic, autoimmune, or inflammatory conditions; malignancies; chronic alcohol use; or were currently on GLP-1 receptor agonists, hepatotoxic drugs, or oral contraceptives.
The primary outcome for the calculation was the between-group difference in mean miR-21 levels. Based on preliminary data (pilot mean difference Δ = 20 units, pooled SD σ = 15), we used the formula for one-way ANOVA sample size (approximated by pairwise two-group comparisons):
$$\:n=\frac{2{(\left({Z}_{1-\frac{{\upalpha\:}}{2}}\:\right)+\left({Z}_{1-\beta\:}\right))}^{2}{{\upsigma\:}}^{2}}{{{\Delta\:}}^{2}}$$
where α = 0.05 (two-tailed), 1 − β = 0.80 (power), Z1−α/2=1.96, Z1−β =0.84. Substituting these values yields ≈ 17 subjects per group.
Allowing for a 15% dropout rate, we planned to recruit at least 20 participants in each group. The final sample sizes (n = 19–45 per group) thus met or exceeded this requirement.
Ophthalmological evaluation
Exclusion criteria encompassed individuals with known ocular diseases other than diabetic retinopathy (DR), a history of ocular surgery, or inflammatory ocular conditions. Participants using topical ocular medications or having other systemic diseases aside from diabetes were also excluded. A thorough ophthalmological examination was performed, including assessments of refractive error, best-corrected visual acuity, slit-lamp examination, intraocular pressure measurement using pneumotonometry, and dilated fundus examination. The severity of diabetic retinopathy was determined using the Early Treatment Diabetic Retinopathy Study (ETDRS) classification.
The evaluation of polyneuropathy
The patients were evaluated for diabetic polyneuropathy by a neurologist using ENMG. According to the reports obtained from ENMG, the patients were found to have polyneuropathy.
Diabetic polyneuropathy was evaluated using electroneuromyography (ENMG). Nerve conduction studies (NCS) were performed bilaterally on the upper and lower limbs while subjects were in a supine position. The Nihon Kohden Neuropack S1 MEB-940 EMG-ER system was used. The median, ulnar, tibial, peroneal, sural, and superficial peroneal nerves were examined for compound muscle action potentials (CMAP) and sensory nerve action potentials (SNAP). Parameters such as onset latency, peak-to-peak amplitude, and conduction velocity were measured and interpreted according to reference values14.
Measurements of carotid intima-media thickness (CIMT)
The extracranial carotid arteries were examined using a standardized protocol by the same radiologist. CIMT was measured using a Siemens Acuson S3000 ultrasound device using a 9L4 (4.0–9.0 MHz) linear transducer. The subjects’ carotid system was evaluated in B-mode, pulsed Doppler mode, and color mode, with the subject in the supine position, with their head slightly turned to the contralateral side of the carotid artery being examined. Carotid IMT is measured by calculating the space between the intimal-luminal and medial-adventitial interfaces of the carotid artery.
Laboratory analyses
Blood samples were obtained at least 24 h before the administration of the drugs, and standardized procedures were followed. Blood specimens were collected following an overnight fast of 10 to 12 h. The serum and plasma samples obtained after centrifugation were stored at −80 °C until adropin analysis.
Levels of serum adropin were assayed by the ELISA kit (Human AD (Adropin), Cat. No. E-EL-H5307, ARP American Research Products, USA). Results were expressed as pg per ml of serum (pg/mL). The sensitivity of this kit was 7.5 ng/L. Intra- and inter-CV were 6.3% and 7.5%, respectively.
Biochemical markers, including glucose, cholesterol, triglycerides, HDL-C, and LDL-C, were assessed using enzymatic techniques on the Roche Cobas Integra 400 analyzer (Germany). Insulin concentrations were determined by electrochemiluminescence immunoassay (ECLIA) using the Roche-Hitachi E170 system. C-reactive protein (CRP) was quantified using nephelometry (Beckman Coulter, Germany), while HbA1c levels were measured by high-performance liquid chromatography (Bio-Rad, Variant Turbo 2, USA). Albumin excretion in 24-hour urine collections was quantified on the Roche Hitachi P800 analyzer. Glomerular filtration rate (GFR) was estimated utilizing the CKD-EPI formula. Insulin resistance was assessed using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), which is calculated from fasting glucose and insulin concentrations according to the following formula:
$$\:HOMA-IR=\:\frac{\text{g}\text{l}\text{u}\text{c}\text{o}\text{s}\text{e}\:\left[\frac{\text{m}\text{g}}{\text{d}\text{L}}\right]\:\times\:\text{i}\text{n}\text{s}\text{u}\text{l}\text{i}\text{n}\:\left[\frac{{\upmu\:}\text{U}}{mL}\right]}{405\:}$$
MiRNA analysis
The total RNA extraction process, inclusive of small RNA from serum, was conducted utilizing the mirVana RNA Isolation Kit (miRNeasy Kit, Qiagen, CA). All isolation procedures were carried out in strict adherence to the manufacturer’s guidelines, with no deviations or modifications made to the standard. The synthesis of complementary DNA (cDNA) was conducted from the total RNA isolated from the serum of all subjects using the miScript Reverse Transcription Kit (Qiagen, Valencia, CA). The concentration and quality of the nucleic acids were determined using the Qubit assay and Qubit fluorometer. The expression levels of hsa-mir-21 (hsa: Homo sapiens) and RNU44 were further quantified using the miScript SYBR® Green PCR Kit (Qiagen, Valencia, CA) in conjunction with miScript-specific primers on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA). RNU44 was chosen as the endogenous control. The relative expression levels of miR-21 were subsequently determined using the 2−ΔΔCT method, with each sample being analyzed in triplicate.
Statistical analysis
SPSS version 22.0 (IBM) was used for statistical evaluations of all data obtained. The normality of continuous variables was assessed using the Shapiro–Wilk test. Variables with a normal distribution are presented as mean ± standard deviation (SD), and those without a normal distribution as median (interquartile range, IQR). Between-group comparisons for normally distributed variables were performed using one-way ANOVA with Bonferroni-adjusted Tukey post-hoc tests; for non-normally distributed variables, the Kruskal–Wallis test was used, with pairwise comparisons by the Mann–Whitney U test. Categorical variables were compared by the χ² test or Fisher’s exact test as appropriate. Correlations were analyzed using Pearson’s or Spearman’s correlation coefficients, depending on normality. To adjust for potential confounders, we constructed multiple linear regression models with circulating miR-21 and adropin levels as dependent variables. As covariates, we included age, sex, body mass index (BMI), hypertension status, smoking status, total cholesterol, and triglyceride levels—factors known to influence vascular biomarkers. We entered all covariates simultaneously (“enter” method) to estimate the independent association of each variable. Before model fitting, we evaluated multicollinearity among independent variables by calculating variance inflation factors (VIFs). All VIFs were < 2.0, indicating no substantial multicollinearity. Receiver operating characteristic (ROC) curve analyses were conducted to determine sensitivity, specificity, area under the curve (AUC), and optimal cut-off values. A two-tailed p-value < 0.05 was considered statistically significant.
