Apelin-13 serum levels in type 2 diabetic obese women: possible relations with microRNAs-107 and 375

Subjects

In this study, among the individuals referred to Massoud medical laboratory in Tehran, Iran. Fifty obese women with newly diagnosed T2D, without any medication, and fifty non-diabetic obese women, as control group, were selected. Exclusion criteria in the present study were obese women with T2D who were treated with medication, women with type 1 diabetes mellitus, hormonal disorders, infectious malady and other diseases that might affect the test results. Inclusion criterion: obese women with newly diagnosed T2D, without taking any medicine. In this work, obesity was determined based on WHO criteria. In adults, obesity is characterized as a body mass index (BMI) ≥30 kg/m². Control group were only obese people and had not any diseases. Diagnostic criteria for T2D, was performed according to WHO standards for symptomatic diabetes mellitus [fasting blood glucose (FBG) ≥126 mg/dL and hemoglobin A1c ≥6.5%]. Protocol of the study was accepted by the Ethics Board of Medical Faculty of Tabriz University of Medical Sciences, on December 8, 2014, with Code number: IR.TBZMED.REC.1393.225. It was based on the Helsinki Declaration. All subjects delivered informed consent before the sampling.

Subject’s information and biochemical measurements

For all subjects, age, height, and weight were recorded and BMI (kg/m²) was also calculated. After 12 h overnight fasting, blood specimens were taken from diabetic and control groups. Specimens were centrifuged and sera aliquoted and reserved at –80°C until assays were FBG was evaluated by enzymatic method (Parsazmun, Karaj, Iran). Determination of glycated hemoglobin or hemoglobin A1c (HbA1c) levels performed by high-performance liquid chromatography (HPLC; DS5 Pink Reagent kit; Drew Scientific, Miami Lakes, FL, USA). The value of estimated average glucose (eAG) was also calculated [19]. High-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum total cholesterol and triglyceride (TG) levels were measured by direct enzymatic procedures (Parsazmun, Karaj, Iran). Apelin-13 levels were assessed with commercial human ELISA kit (Korain Biotech Company, Shanghai TECH, China), with sensitivity 0.27 ng/L and a within (intra) and between (inter) assay coefficient of variation (CV) of 8% and 10%, respectively. Insulin (with an intra- and inter-assay CV of 5.1–8.3% and 7.2–11.3%, respectively), estradiol or E2 (with an intra- and inter-assay CV of 7.5–9.9% and 4.8–8.2%, respectively) and progesterone (with an intra- and inter-assay CV of 6.1–15.3% and 6.4–8.9%, respectively) levels were measured by ELISA (Monobind Inc. Lake Forest, CA, USA). Serotonin (with an intra- and inter-assay CV of 4.2–11% and 12.7–16.2%, respectively) levels were measured by ELISA (Abcam Inc., Cambridge, MA, USA).

eAG values were reported along with HbA1c, including the fact that it can be considered as a potential factor in T2D follow up. eAG values for each group were calculated according to the following formula:

eAG (mg/dL)=HbA1c×28.7−46.7

Quantitative real-time PCR

The expression levels of hsa-miR-107 (target sequence 5′-AGCAGCAUUGUACAGGGCUAUCA-3′) and hsa-miR-375 (target sequence 5′-UUUGUUCGUUCGGCUCGCGUGA-3′) in serum of diabetic and control subjects were measured by real-time PCR (qPCR). The primers (1 pg), miRCURY LNA™ Universal RT microRNA PCR, LNA™ primers set, were purchased (Exiqon, Denmark) in a ready-designed form to detection of target microRNA. Total microRNA was extracted utilizing exiqon miRCURY RNA extraction kit-biofluids (Exiqon, Denmark), following the recommended protocol. Concentration and purity of the extracted RNA was evaluated by NanoDrop 1000 spectrophotometer V3.7 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared utilizing LNA universal RT miRNA PCR kit (Exiqon, Vedbæk, Denmark), as specified by the manufacturer. The reverse transcriptase reaction was performed by incubations at 42°C for 60 min, 95°C for 5 min and then immediate cooling at 4°C using an Eppendorf thermal cycler (Hamburg, Germany). Five microlitre from synthesized cDNA (20 ng), was diluted with 395 μL nuclease free water. Quantification of microRNAs was performed using SYBR Green master mix with LNA-based miRNA primers (Exiqon, Vedbæk, Denmark). For real-time PCR, the reaction mixture consisted of 5 μL PCR master mix, 1 μL primer mix and various volumes of diluted cDNA sample was performed by incubating at 95°C for 10 min followed by 45 cycles at 95°C for 10 s and 63°C for 1 min, ramp rate 1.6°C/s; followed by a melting at 60–95°C using the mic real-time PCR instrument (Bio Molecular Systems, Upper Coomera, Australia). miRNA U6 was served as the endogenous reference. The reactions were performed in triplicate. In order to optimizing and verifying the specificity of amplified target genes, we performed a real-time-PCR amplification of serially diluted cDNA (in triplicate) and then we assayed amplification curves and melting curves.

Cell culture

CRI-D2 cell lines, with C187 NCBI code, were purchased Pasteur Institute (Tehran, Iran). CRI-D2 is derived from the islands of Langerhans of a NEDH rat transplantable islet cell tumor. Cells secrete insulin and glucagon. Cells were cultivated in RPMI-1640 medium (Gibco, Invitrogen), accompanied with 10% fetal bovine serum (FBS) and preserved in 5% of CO₂ at 37°C. After five times passage, the cells were seeded in six-well culture plates. Our groups were including untreated, as control, and six groups treated with 50 nmol/L and 100 nmol/L apelin-13 trifluoroacetate salt (Sigma, , St. Louis, MO, USA) in different hours (24, 48, and 72 h). Expression of miR-107, miR-375 and miRNA U6 were assayed, as mentioned in qPCR section. Insulin levels in cells media were determined with commercial rat ELISA kit, with an intra- and inter-assay precision CV ≤10.0% (Crystal Chem Incorporated, Downers Grove, IL, USA).

Flow cytometry analysis for cell apoptosis

To study apoptosis in the CRI-D2 cell line, flow cytometry analysis was performed. After incubation of control and apelin-13-treated groups for 24, 48 and 72 h, the cells were prepared for flow cytometry analysis using the ApoFlowEx FITC Kit (EXBIO Praha, Czech Republic), following the manufacturer’s recommended procedure for flow cytometer analysis (BD FACSCalibur, USA).

Statistical analysis

Statistical analysis was accomplished utilizing SPSS.16 (IBM, USA) software. The outcomes were shown as mean±SD. The expression levels of miRNAs were computed by utilizing the ΔCt method. All of the data had normal distribution; statistical analysis was done by student-t test. The Pearson correlation test was used to analyze the relationships between variables, a two-tailed p-value of <0.05 and <0.01 were accepted as statistically significant. For statistical analysis of microRNAs expression levels and insulin concentrations in cell culture, we used one-way ANOVA. After one-way ANOVA test, tukey post hoc was used. In all analyzes, a p-value less than 0.05 characterized the presence of a statistically substantial difference.

Distribution of the general and the metabolic characteristics

We studied the general and the metabolic variables in diabetic and non-diabetic individuals. All of the parameters, with the exception of HDL-C (p-value=0.22), weight (p-value=0.1) and height (p-value=0.3) were considerable in the mentioned subjects (Table 1).

Comparison and correlation between the Apelin-13 and insulin, estradiol, progesterone, and serotonin in groups

We assayed insulin, estradiol, progesterone, serotonin, and Apelin-13 in the diabetic and non-diabetic groups. Significant results were observed in the levels of insulin, estradiol, progesterone, serotonin (p<0.001) and apelin-13 (p=0.012) in the diabetic group compared with control group (Table 1). The homeostatic model assessment of insulin resistance (HOMA-IR) was utilized to evaluating of insulin sensitivity. Because our study groups were female obese subjects and in menopausal status, we measured sexual hormones (estradiol and progesterone). There was no correlation between apelin-13 and miRs (miR-107 and miR-375) in two groups. There was a very weak negative correlation between Apelin-13 and FBG, TG, HA1c and BMI. Correlation between apelin-13 and HOMA-IR was very weak positive. In the diabetic group, correlation between apelin-13 and serotonin (p<0.001), eAG (p<0.02) and insulin (p<0.03) statistically was significant (Table 2). There is no correlation between apelin and the above-mentioned parameters in the control group.

High expression of miR-107 and miR-375 in diabetic subjects

The levels of circulating miR-107 and miR-375 were determined in the sera samples of diabetic and non-diabetic subjects. Results showed that the expression levels in both miRs in diabetic group were significantly greater versus non-diabetic group (p-value=0.02). Fold changes in the levels of miR-107 and miR-375 were calculated with ΔCt method using U6 as internal reference miRNA. The fold change value of these microRNAs has been shown as mean value. The mean value of samples for non-diabetic group aligned to a value of 1.0 and the all diabetic sample values were declared concerning this aligned mean value. (Figure 1A and B).

Figure 1:

(A and B) microRNA-107 (left) and microRNA-375 (right) fold changes by quantitative PCR (qPCR) analysis in serum of diabetic and control groups.

The fold change value for each miRNA is expressed by mean value. For diabetic group, data is shown as mean±SD. For control group data is shown as mean. *Significance versus control group. p<0.05.

Insulin and microRNAs expression in cultured CRI-D2 cell lines

In this work, we applied the insulinoma cell line (CRI-D2) as a pure beta cells source, while islets are a complex of different cells which secrete different hormones. This cell line cultured in six-well culture plates, as previously was explained, thereafter, they were treated with apelin-13 to determine insulin levels and microRNAs expression in response to apelin-13 stimulation. Our data demonstrated that apelin-13 has no significant effect on levels of insulin (in hours and different concentrations of incubation with apelin-13) (Figure 2). Our data showed a high expression of miR-107 (p<0.05) in six groups and miR-375 (p<0.05 and <0.001) (Figure 3A and B, respectively).

Figure 2:

Levels of insulin in medium of CRI-D2 cell culture treated with apelin-13 trifluoroacetate salt.

Data are shown as mean±SD.

Figure 3:

(A and B) Relative expression miR-107 (left) and miR-375 (right) in medium of CRI-D2 cell culture treated with apelin-13 trifluoroacetate salt.

Data are shown as mean±SD. *Significance versus control group. p<0.05, **significance versus other groups and control group. p<0.001.

Apelin-13 has no effect on CRI-D2 cell line apoptosis

After the incubation of CRI-D2 cell line in hours and different concentrations of apelin-13, apoptosis (by flow cytometry technique) was assayed. Our data showed apelin-13 has no effect on apoptosis and thereupon, cell death (Figure 4).

Figure 4:

Flow cytometry assay data, cells were treated with 0, 50, and 100 nM of apelin-13 in different hours (24, 48, and 72 h).

Quadrants represents, Q3: viable cells, Q4: apoptotic cells, and Q2: late apoptotic or necrotic cells (according to the kits instructions). It was not observed any apoptosis in the groups.