Increased epicardial adipose tissue in type 1 diabetes is associated with central obesity and metabolic syndrome

Methods Forty-five type 1 diabetic women were evaluated (age 36 ± 9 years; body mass index 24.6 ± 4.4 kg/m 2 ). Metabolic syndrome was defined by the World Health Organization criteria. Body fat composition and EAT were analyzed by dual-energy-X-ray absorptiometry and echocardiogram, respectively. Results Twenty patients (45%) had MS. Patients with MS had greater android (central) fat deposition than patients without MS (41.9 ± 2.0% vs . 33.7 ± 1.8%, p = 0.004). Total body fat and gynoid (peripheric) fat distribution were similar between the groups. Mean EAT was higher in patients with MS (6.15 ± 0.34 mm vs . 4.96 ± 0.25 mm; p = 0.006) and EAT was positively correlated with android (central) fat distribution ( r = 0.44; p = 0.002), however no correlation was found with gynoid (peripheric) fat distribution. Conclusions There was a high incidence of MS in type 1 diabetes related to increased central adiposity, despite the absence of obesity. Metabolic syndrome and central obesity were associated with increased EAT. Thus, young non-obese type 1 diabetic women with central adiposity and/or MS may have increased EAT, what may predict CAD risk. Keywords Type 1 diabetes Epicardial adipose tissue Metabolic syndrome Body fat composition 1 Introduction It is well established that type 1 diabetes mellitus (DM) is associated with a high incidence of cardiovascular disease (CVD) [1] and early mortality [2,3] . A great number of CVD risk factors accumulate in type 1 DM persons in the early age. Women with type 1 DM experience greater relative risks of CVD than men compared with those without diabetes, especially in young ages [2,3] . In addition, more young people today are overweight and obese, and the development of insulin resistance and metabolic syndrome (MS) with its associated atherogenic risk factors in type 1 DM may further increase the CVD risk [4] . The MS has been shown to predict major complications outcomes in type 1 DM, including coronary artery disease, renal failure and diabetes related death in several clinical studies [5–8] . On the contrary, in other study the presence of MS did not add significant prognostic predictive value to conventional vascular risk factors [9] . The real impact of MS in type 1 DM still needs to be further evaluated. The MS is characterized by the clustering of independent CVD risk factors including insulin resistance, central obesity, impaired glucose metabolism, hypertension and dyslipidemia [10–13] . The evaluation of body fat distribution is important to identify patients at risk of MS, since visceral obesity appears to play a key role in the development of the MS [14,15] . Epicardial adipose tissue (EAT) is a particular form of visceral adipose tissue deposited around the heart and the subepicardial coronary arteries. Epicardial fat measured by echocardiography has been associated with visceral obesity [16–19] and has emerged as a new independent predictor of coronary artery disease [20–25] . Epicardial fat thickness was correlated with the presence and severity of coronary artery disease in several clinical settings [20–25] . In type 2 DM patients, an increased EAT volume was associated MS and coronary atherosclerosis [26] . There is no report on the literature describing EAT volume in type 1 DM. Epicardial adipose tissue measured by transthoracic echocardiography is a validated method, easily performed and with a favorable cost-benefit, that could be used to predict coronary artery disease in type 1 DM. Similarly, body fat composition analysis and evaluation of MS parameters may give additional information about the cardio-metabolic risk in the clinical practice. In the present study, young women with type 1 DM were evaluated for the presence of MS clinical parameters and for the analysis of body fat distribution and EAT. The aim of this study was to determine the relationship between EAT, body fat composition and MS in type 1 women with DM. We hypothesized that type 1 DM women with MS and central obesity would have increased EAT deposition. 2 Subjects The study group consisted of women with type 1 DM randomly recruited from the outpatient diabetes clinic of Diabetes and Endocrinology State Institute, IEDE, over a 6 months period. A total of 45 women were enrolled. All subjects entered the study after written informed consent according to a protocol approved by the local ethics committee. Inclusion criteria were continuous insulin therapy since the diagnosis and the presence of positive anti-glutamic acid decarboxilase (GAD) auto-antibodies. Reasons for ineligibility included past history of cardiovascular disease and ovarian failure. After evaluation of MS clinical parameters, patients were divided in two groups according to the presence of MS: (1) Type 1 diabetes with MS; (2) Type 1 diabetes without MS. 2.1 Definition of metabolic syndrome Metabolic syndrome was defined according to the World Health Organization (WHO) consensus criteria [12] . The WHO definition requires a presence of glucose intolerance or diabetes and/ or insulin resistance for diagnosis and two of the following: (1) hypertension, defined as antihypertensive treatment and/or elevated blood pressure (≥160 mmHg systolic or ≥90 mmHg diastolic); (2) dyslipidemia, defined as elevated plasma triglycerides (≥150 mg/dl) and/or low HDL cholesterol (≤35 mg/dl in women); (3) obesity, defined as high BMI (>30 kg/m 2 ) and/or high waist-to-hip ratio (WHR) (≥0.85 in women); (4) microalbuminuria (urine albumin excretion rate ≥20 mcg/min). 3 Materials and methods 3.1 Anthropometric parameters Weight (in kilograms-kg) and height (in meters-m) were measured with the subjects wearing only their undergarments. Body mass index (BMI) was calculated as body weight divided by height squared (kg/m 2 ). Waist (WC) and hip circumferences (in centimeters-cm) were measured in the midline between the lower rib margin and the iliac crest, and widest diameter over the greater trochanters, respectively, while the subjects were standing with their hells together. Waist-to-hip ratio (WHR) was obtained. 3.2 Laboratory measurements Fasting blood samples were collected and analyzed for glycated hemoglobin (HbA1c) and lipids. HbA1c concentrations were measured by high-pressure liquid chromatography with a reference range of 4–6% (Variant II, Biorad). Serum total cholesterol, HDL cholesterol and triglycerides were measured by a calorimetric enzymatic assay (Advia, Siemens). LDL cholesterol was calculated using the Friedewald formula [27] . Microalbuminuria (urine albumin excretion rate) was analyzed in a 24 h urine sample and was determined by nefelometric method (BNII, Siemens). 3.3 Dual-energy-X-ray-absorptiometry (DXA) Total body dual-energy-X-ray-absorptiometry was performed using a GE Lunar Prodigy Advance scanner (software 11.2, GE ® Healthcare, Belgium). Total body and regional body fat composition were analyzed. Regional fat distribution is measured by DXA as android and gynoid fat regions. Total body fat (TBF), android and gynoid fat regions were expressed as a percentage of total body weight. The android to gynoid fat ratio (A/G) was also determined. Android fat region is an estimative of central fat, while gynoid fat region correlates with peripheral fat. Android fat region has been shown to contain a relative high proportion of intra-abdominal fat and has been validated as a good indirect method of visceral fat prediction [28–33] . 3.4 Echocardiographic study Each patient underwent a complete transthoracic echocardiography using the American Society of Echocardiography guidelines of measurement. Echocardiograms were performed with a VIVID 7 (GE, USA) instrument according to standard techniques, using a M3S transducer, with subjects in the left lateral decubitus position. Echocardiographic images were performed by the same cardiologist with high experience in echocardiography, who was unaware of the clinical data. Images were recorded into a computerized database. The EAT thickness was measured on the free wall of the right ventricle from the paraesternal long-axis views, as previously described and validated [17] . Epicardial adipose tissue was identified as an echo-free space in the pericardial layers on the 2-dimensional echocardiography, and its thickness was measured perpendicularly on the free wall of the right ventricle at end-diastole (QRS complex) for 3 cardiac cycles. The measurement was performed at a point on the free wall of the right ventricle along the midline of the ultrasound beam, perpendicular to the aortic annulus ( Fig. 1 ). In order to increase confidence in the results, EAT measurement by echocardiogram was performed in two different moments. The results found in the two measurements were significantly correlated, with a reliability of 92.4% ( p < 0.001). 3.5 Statistical analysis Statistical analysis was performed using the program GraphPad Prism ® (version 4.00 for Windows, GraphPad Software, San Diego, CA, USA). Data were expressed as mean ± standard deviation (SD). Patient́s baseline characteristics, body fat composition and epicardial adipose tissue in the two groups (type 1 diabetes with and without MS) were compared using an independent Student's t -test. The correlation between the parameters was tested by Pearson correlation. Statistical significance was set at p < 0.05. 4 Results Subjects in the study were female with a mean age of 36 ± 9 years, mean diabetes duration of 18 ± 9 years and mean BMI of 24.6 ± 4.4 kg/m 2 . Using the WHO criteria, 20 type 1 diabetic patients had MS. The prevalence of MS in this population was of 45%. Table 1 shows the clinical characteristics of type 1 diabetic patients with and without MS. None of the subjects in the study had obesity, defined by a BMI > 30 kg/m 2 . Mean BMI was 26.6 ± 0.9 kg/m 2 in patients with MS and 23.0 ± 0.8 kg/m 2 in patients without MS ( p = 0.0004). There were no age or race differences between the groups. Patients with and without MS had similar type 1 DM mean duration in years. Glycemic control was also similar between patients with and without MS, since mean HbA1c were not statistically different. Body composition analysis by DXA ( Table 2 ) demonstrated an increased central fat deposition in the group of type 1 DM patients with MS, with a higher android fat distribution and A/G ratio than patients without MS (41.9 ± 2.0% vs . 33.7 ± 1.8%, p = 0.004; and 0.94 ± 0.05 vs . 0.73 ± 0.03, p = 0.002). Total body fat and gynoid fat distribution were not different between the groups with and without MS (38.4 ± 1.8% vs . 35.4 ± 1.4%, p = 0.19; and 45.1 ± 1.8% vs . 45.9 ± 1.2%, p = 0.71; respectively) ( Table 2 ). Furthermore, patients with type 1 DM and MS also had increased WHR ( Table 1 ). Waist-to-hip ratio was positively correlated with TBF ( r = 0.33; 95% confidence interval-95CI = 0.07–0.56; p = 0.03), android fat deposition ( r = 0.56; 95CI = 0.31–0.73; p < 0.0001) and A/G ratio ( r = 0.70; 95CI = 0.51–0.82; p < 0.0001); but not with gynoid fat deposition ( r = −0.006; 95CI = −0.30–0.29; p = 0.9). Android fat deposition was associated with increased LDL cholesterol ( r = 0.38; 95CI = 0.09–0.60; p = 0.01), increased triglycerides ( r = 0.35; 95CI = 0.06–0.59; p = 0.019) and decreased HDL cholesterol ( r = −0.38; 95CI = −0.60 to −0.09; p = 0.01). Similarly, A/G negatively correlated with HDL cholesterol ( r = −0.45; 95CI = −0.66 to −0.18; p = 0.002). Patients with hypertension ( n = 15) had higher A/G ratio than patients without hypertension ( n = 30) (0.91 ± 0.06 vs . 0.78 ± 0.03; p = 0.02). Epicardial adipose tissue was increased in type 1 DM subjects with MS ( Fig. 2 ). Mean EAT was higher in patients with MS than in patients without MS (6.15 ± 0.34 mm vs . 4.96 ± 0.25 mm, respectively; p = 0.006). Patients with hypertension ( n = 15) also had a greater EAT volume than patients without hypertension ( n = 30) (6.2 ± 0.45 mm vs . 5.13 ± 0.23 mm, respectively; p = 0.02). Epicardial adipose tissue was positively correlated with android fat distribution ( r = 0.44; IC95 =0.17–0.65; p = 0.002) ( Fig. 3 ), with TBF ( r = 0.43; 95CI = 0.16–0.64; p = 0.003), with A/G ratio ( r = 0.43; 95CI = 0.16–0.64; p = 0.003) and with WHR ( r = 0.57; 95CI = 0.33–0.74; p < 0.001); but there was no correlation between EAT and gynoid fat deposition ( r = 0.27; 95CI = −0.03 to 0.52; p = 0.07). There was also no association of EAT deposition with duration of type 1 DM ( r = 0.27; 95CI = −0.03 to 0.52; p = 0.75) or glycemic control measured by mean HbA1c ( r = −0.06; 95CI = −0.35 to 0.24; p = 0.69). Microalbuminuria was increased in patients with type 1 DM with MS compared with patients without MS (22.70 ± 7.38 mcg/min vs . 8.12 ± 0.78 mcg/min; p = 0.03) ( Table 1 ). Nevertheless, microalbuminuria was not correlated with TBF ( r = −0.09; 95CI = −0.37 to 0.20; p = 0.54), android fat ( r = 0.18; 95CI = −0.31 to 0.28; p = 0.90) nor gynoid fat deposition ( r = −0.13; 95CI = −0.41 to 0.17; p = 0.4). Epicardial adipose tissue deposition was also not associated with microalbuminuria ( r = 0.04; 95CI = −0.26 to 0.33; p = 0.79). 5 Discussion To our knowledge, the present study is the first that describes EAT in women with type 1 DM. In our study, clinical parameters of MS and central adiposity were associated with increased EAT, what may suggest higher cardiovascular risk in this population. We evaluated young non-obese type 1 DM women, with no previous history of cardiovascular disease (CVD) and with normal ovarian function. This population was selected based on previous data demonstrating that young type 1 DM women experience greater relative risks of CVD and an exceptionally high mortality risk from ischemic heart disease compared to men with DM and to the general population without DM, especially under the age of 40 years old [2,3] . Therefore, it is important to determine in this population factors that may represent an additional CVD risk. We also included only women with normal ovarian function. Our purpose was to avoid any bias caused by the presence of early menopause or polycystic ovarian syndrome. There is evidence in the literature that these ovary dysfunctions may be related to increased CVD risk [34–36] and to altered body fat composition, with increased central fat [35–39] . Previous reports have described that the association between type 1 DM and MS, the so called “double diabetes”, was related to coronary artery disease and DM related death [5,6] . The WHO criteria of MS appear to have the highest sensitivity to discriminate negative outcomes in patients with type 1 DM [6,40] . There was a high prevalence of MS in our study group of 45%, using the WHO criteria. Prior studies suggested that the MS is a frequent finding in type 1 DM [5–7,40] . In a multicenter study involving 2.415 type 1 DM patients the overall prevalence of MS was of 40% in women [5] , which corroborates our data. Type 1 DM women with MS presented with an increased central fat distribution. Using DXA, we found that patients with MS had an increased android fat distribution and a mean android fat of 41.9% compared with a mean of 33.7% in patients without MS. Total body fat and gynoid (peripheric) fat distribution did not differ in patients with and without MS ( Table 2 ). Similarly, patients with MS had increased WHR ( Table 1 ), which was positively correlated with android fat deposition measured by DXA. It has been demonstrated that central obesity is associated with an increased cardiovascular risk and with MS [10,14,15,41] . Dual-energy-X-ray absorptiometry has been validated as a precise method for body fat evaluation and an indirect method of visceral fat determination. Regional fat distribution measured by DXA as gynoid fat and android fat positively correlates with central and peripheral fat measured by magnetic resonance, respectively [28–33] . An important feature of our study is that subjects analyzed had normal weight or were overweigh, but none had obesity, given that the mean BMI was of 24.6 ± 4.4 kg/m 2 . Our data reinforce the importance of body fat composition analysis in type 1 DM and demonstrates that even non-obese type 1 DM women might have increased cardio-MS risk, if there is a predominant deposition of central fat. In the present study, type 1 DM patients with MS and/or central obesity had increased EAT measured by echocardiography. Epicardial adipose tissue deposition was associated with clinical and anthropometric parameters of the MS ( Fig. 2 ). We also observed an association between EAT and central obesity, as there was a significant correlation of EAT with android fat distribution and A/G ratio measured by DXA ( Fig. 3 ) and with WHR. No correlation was found of EAT with peripheric fat deposition, represented by the gynoid fat region. Poor glycemic control or DM duration did not seem to influence EAT deposition in this study. Our data suggest that body fat distribution, particularly central fat tissue, is more strongly correlated with EAT. Prior studies have demonstrated in non-diabetic individuals that EAT was associated with central obesity [17–19] , clinical parameters of the MS [16,26] and insulin resistance [42] . Hypertension was also related to increased EAT in type 1 DM in our population, irrespective of the presence of MS. Nonetheless, hypertension was present in a great proportion of patients with MS and central obesity. It is interest to notice that central obesity is an important feature in the pathophysiology of both hypertension and MS [10,14,41] . Therefore, our study suggests that the presence of three inter-related risk factors might be associated with increased EAT: central obesity, MS and HAS. Chronic complication of type 1 DM represented by nephropathy did not seem to influence EAT deposition. We found no correlation of microalbuminuria with EAT in type 1 DM patients with and without MS. To our knowledge, this was the first study to address this issue. Microalbuminiria was, however, more prevalent in patients with MS and type 1 DM. This was expected given that we used the WHO criteria to discriminate the presence of MS, which includes microabuminuria in the definition of MS. Previous studies also described association of MS with advanced nephropathy in type 1 DM [5,6] . Interestingly, we found that the presence of microalbuminuria was not influenced by body fat composition. The finding of elevated an EAT in type 1 DM with MS may contribute to an unfavorable cardiovascular profile observed in these patients. In previous studies, echocardiographic EAT thickness was associated with the severity of coronary artery stenosis assessed by coronary angiograms [21–26] . Indeed, EAT may play a role in the development of coronary atherosclerosis via the association with conventional risk factors and also through direct endocrine and paracrine effects [24,43,44] . It has been demonstrated that it is a source of pro-inflammatory cytokines such as nuclear factor-KB and c-jun n-terminal kinase that may promote local and paracrine inflammation [43,44] . Echocardiography was chosen as the method of EAT measurement in this study because it is an easy, non-invasive, cost effective, widespread method of cardiovascular evaluation in diabetic patients. We measured EAT as previously validated by the studies of Iacobellis and col. They demonstrated that EAT measured by transthoracic echocardiography with the correct technique may have results comparable to that obtained with magnetic resonance imaging [16,17] , with the advantage of not exposing patients to radiation or to contrast media. There is still no threshold value established of high-risk echocardiographic EAT thickness [45] . In the present study, type 1 DM women with MS had a mean EAT of 6.15 ± 0.34 mm. Anh et al., demonstrated that EAT thickness over 3 mm was an independent factor for CVD [22] . In the study of Jeong et al., the incidence of significant coronary stenosis increased in proportion to the EAT thickness, with the higher risk observed over 7.6 mm [20] . In patients with known coronary artery disease it has been suggested that the higher risk threshold value in women should be of 7.5 mm [24] . Our population was of young subjects with no previous history of cardiovascular disease, so we would not expect an elevated EAT thickness. Nevertheless, we should consider the increasing rate of EAT thickness in patients with type 1 DM and MS as an risk factor for coronary artery disease. The cross-sectional study design confers several limitations. First, it is not possible to accurately determine a causal relationship between EAT volume, central obesity and MS with the measurement of these parameters in a single time period. Furthermore, we could not directly assess the incidence of cardiovascular disease in type 1 DM with MS. Nevertheless, since EAT volume has been associated with coronary artery disease in general population, we could infer that a higher EAT volume in type 1 DM women with MS could be related with increased cardiovascular disease. Prospective studies with prolonged follow-up are necessary to better evaluate cardiovascular outcomes in patients with type 1 DM, MS and increased EAT. In conclusion, we observed a high incidence of MS in type 1 DM that was related to increased central adiposity, despite the absence of obesity. Metabolic syndrome, central obesity and hypertension were associated with increased EAT, which has emerged as a new marker of coronary artery disease. Thus, our study provides evidence that non-obese young adult women with type 1 DM that exhibit central adiposity and/or clinical parameters of the MS may have increased EAT. These patients may benefit of further cardiovascular evaluation to assess the presence of coronary artery disease, with the purpose of preventing CVD morbidity and mortality. Conflicts of interest statement Authors disclose no conflicts of interest regarding this manuscript. Acknowledgements The authors thank Dra Giovanna A. 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