Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

A total of 44 patients who underwent coronary artery bypass grafting CABG (CAD group, 38 men and six women) and 23 patients who underwent aortic and/or mitral valve replacement (patients without CAD (NCAD), 12 men and 11 women) were enrolled in this study. The CAD group was divided into two groups: those with DM2 (CAD-DM2, 18 men and four women) and those without DM2 (CAD-NDM2, 20 men and two women). The CAD group was defined by the presence of at least one coronary stenosis ≥50 % of luminal diameter by coronary angiogram. The NCAD group (12 men and 11 women) had chronic valvular heart disease with or without stenosis less than 50 % in any vessel requiring valve replacement but not CABG and without DM2. Exclusion criteria were acute inflammatory disease, severe infective disease and/or cancer, and women who were taking hormone replacement therapy. DM2 was defined as having a history of diabetes diagnosed and/or treated with medication, fasting blood glucose ≥126 mg/dl and/or glycated hemoglobin (HbA1c) ≥6.5. All patients gave written informed consent, and the study was reviewed and approved by the Ethics and Research Committee of Virgen de la Victoria Clinical University Hospital, Malaga, Spain, and carried out in accordance with the Declaration of Helsinki.

Human EAT biopsy samples (average 0.2–0.5 g) were taken near the proximal right coronary artery and SAT (average 1.5–2 g) was obtained from the thorax. Thoracic SAT biopsies were taken 30–45 min after anesthesia and EAT biopsies were taken approximately 1 h after anesthesia. All the tissues were frozen immediately in liquid nitrogen and stored at −80 °C for RNA isolation.

Adipose tissue samples were minced in TriZol reagent (Invitrogen) and homogenized completely on ice. Total RNA was extracted by chloroform and purified through RNeasy minicolumns. After on-column DNase treatment, RNA was eluted with Rnase-free water. Total RNA was quantified with a spectrophotometer (Nanodrop N-100, Thermo Scientific), and all samples had a 260/280 nm absorbance ratio ≥1.8. Reverse transcriptions were performed using 1 μg of total RNA with Transcriptor First Strand cDNA Synthesis Kit (Roche) and random hexamers in 20 μl reaction. The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)]. Real-time PCR amplifications were performed on 96-well plates in reaction buffer containing Taqman Universal PCR Master Mix (No AmpErase UNG, Applied Biosystems, USA), 150 nM Taqman probe, 900 nM primers, and 22.5 ng cDNA. PCR reaction conditions were 48 °C for 30 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min using an ABI 7500 Fast Detection System (Applied Biosystems). Data were obtained as Ct values according to the manufacturer’s guidelines (the cycle number at which logarithmic PCR plots cross a calculated threshold line) and were used to determine ΔCt values (ΔCt = Ct of the target gene minus Ct of the housekeeping gene). Cyclophilin A transcripts were amplified in the same reaction to normalize for variance in input RNA. mRNA expression levels relative to cyclophilin A were calculated by the 2^−ΔCt method. All tests were performed in duplicate. A negative control, RNA amplification without previous retrotranscription, was done to test for possible genomic DNA contamination.

Normality of continuous variables was checked by means of the Kolmogorov–Smirnov test. Continuous variables are expressed as mean ± standard deviation, and as the mean ± SEM in figures and compared by the use of analysis of variance and the post hoc analysis Bonferroni test or with the Student’s test. Differences between EAT and SAT were analyzed with the paired Student’s t test. Categorical variables expressed as proportion and compared by use of Chi squared test. The Pearson correlation coefficient was calculated to estimate the linear correlations between variables. Independent predictors of EAT PGC1α mRNA levels were determined by multiple regression analysis and variables achieving P < 0.05 on correlation analysis were included. To define the risk factors of the development of coronary lesions, multiple logistic regression analysis was used and odds ratios (OR) were calculated. The inclusion of 3 patients in each group, for a a-value of 0.05, (assuming a difference of 1.28 and a standard deviation of 0.28), has the power to detect a significance difference of 95 %. Statistical analyses were performed with SPSS for Windows version 15 (SPSS Inc. Chicago, IL, USA). Values were considered to be statistically significant when P < 0.05.

The general characteristics of the patients in the three groups are summarized in Table 1. No differences between the group of CAD patients with and without DM2 were found for either established risk factors, consumption of medications or general clinical characteristics. Diabetic treatment of CAD patients with DM2 was: only diet (n = 4, 19 %), oral anti-diabetic (n = 12, 57.1 %), oral anti-diabetic and insulin (n = 4, 19 %) and only insulin (n = 1, 4.9 %). No patient was taking thiazolidinediones. Percentage of CAD-DM2 patients on oral-antidiabetic were: metformin, 73 %, sulfonylureas (gliclazide and glibenclamide), 23 %.

To investigate possible differential expression between the two groups of CAD patients (DM2 and NDM2) and NCAD, mRNA expression levels of EAT and SAT brown fat genes were evaluated. Values are gives as fold increase relative to the NCAD group and differences between groups were considered significant at P < 0.05 (Fig. 1).

PGC1α and UCP1 mRNAs in EAT were significantly lower in the CAD-DM2 patients compared with the CAD-NDM2 patients (P = 0.027 and P = 0.038, respectively) and NCAD patients (p = 0.031 and p = 0.045, respectively) (Fig. 1a, b). However, in contrast to EAT, there was a trend for higher PGC1α and UCP1 mRNA expression levels in the SAT from the CAD-DM2 patients compared with the CAD-NDM2 patients, though it was not statistically significant (Fig. 1d, e). PRDM16 mRNA expression was lower in the EAT (Fig. 1c) and higher in the SAT (Fig. 1f) from the CAD group compared with NCAD, but it was not statistically significant.

To elucidate potential pathophysiological implications in gene expression, we compared the expression profile of the target genes involved in thermogenic and mitochondrial function in the EAT and SAT of patients with CAD (Fig. 2).

PGC1α mRNA levels were significantly higher (1.6-fold) in thoracic SAT that in EAT (P = 0.0029). Conversely, UCP1 mRNA levels were significantly lower (13-fold) in thoracic SAT than in EAT (P < 0.0001). PRDM16 mRNA was similar in EAT and SAT.

The PGC1α mRNA expression levels in EAT correlated positively with HDL cholesterol (r = 0.498, P = 0.004), LVEF (r = 0.376, P = 0.014), and EAT UCP1 mRNA expression (r = 0.307, P = 0.043) and negatively with the BMI (r = −0.431, P = 0.025), serum levels of triglycerides (r = −0.468, P = 0.005). Indeed, EAT PGC1α mRNA expression levels differed with the number of injured coronary arteries (P = 0.0005) (Fig. 3). Thus, PGC1α mRNA expression was higher in patients with one or two injured coronary arteries (2^−ΔCt = 0.0038 ± 0.0014) than in those with three or left main trunk injured coronary arteries (2^−ΔCt = 0.0023 ± 0.0013). Considering only men, EAT PGC1α mRNA expression levels with the number of injured vessels remained statistically significant (P = 0.020). Multiple regression analysis showed that BMI (β = −0.081, P = 0.048; 95 % CI −0.162, −0.001), LVEF (β = 0.043, P = 0.006, 95 % CI 0.013, 0.072), age (β = 0.072, P = 0.003, 95 % CI 0.026, 0.117), EAT UCP1 mRNA levels (β = 0.148, P = 0.001, 95 % CI 0.063, 0.233) and DM2 (β = −0.929, P = 0.044, 95 % CI −1.832, −0.026) were independently associated with EAT PGC1α mRNA levels (Table 2). Finally, logistic regression analysis showed the PGC1α mRNA expression in EAT to be a protective factor against the number of injured coronary arteries (OR = 0.310; P = 0.037, 95 % CI = 0.103, 0.931). However, circulating triglyceride levels were associated with a higher risk of suffering coronary lesions (OR = 1.023; P = 0.044, 95 % CI = 1.001, 1.046; p < 0.05) (Table 3).

All the subjects were patients with valvular heart disease or CAD. Only small samples of EAT were collected from each patient and they were insufficient for a thorough analysis and correlation between mRNA and protein.