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PAPER. Free testosterone plasma levels are negatively associated with the intima-media thickness of the common carotid artery in overweight and obese.
International Journal of Obesity (2003) 27, 803–807 & 2003 Nature Publishing Group All rights reserved 0307-0565/03 $25.00 www.nature.com/ijo

PAPER Free testosterone plasma levels are negatively associated with the intima-media thickness of the common carotid artery in overweight and obese glucose-tolerant young adult men G De Pergola1, N Pannacciulli1, M Ciccone2, M Tartagni3, P Rizzon2 and R Giorgino1 1 Internal Medicine, Endocrinology, and Metabolic Diseases, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy; 2Cardiovascular Diseases, Department of Clinical Methodology and Medical-Surgical Technologies, University of Bari, Bari, Italy; and 3Istituto di Clinica Ginecologica I; University of Bari, Bari, Italy

OBJECTIVE: To evaluate the relation between free testosterone (FT) levels and the intima-media thickness of the common carotid artery (IMT-CCA) in overweight and obese glucose-tolerant (NGT) young adult men. DESIGN: Cross-sectional study of FT and IMT-CCA in obese men. SUBJECTS: A total of 127 overweight and obese NGT male individuals, aged 18–45 y. MEASUREMENTS: FT plasma levels; IMT-CCA, as measured by high-resolution B-mode ultrasound imaging; central fat accumulation, as evaluated by waist circumference; body composition, as measured by bioimpedance analysis; insulin resistance, as calculated by homeostatic model assessment (HOMAIR); systolic and diastolic blood pressure; and fasting concentrations of glucose, insulin, and lipids. RESULTS: IMT-CCA was positively correlated with age, body mass index (BMI), fat mass (FM), waist circumference, and fasting glucose concentrations, and inversely associated with FT levels. After multivariate analysis, IMT-CCA maintained an independent association with BMI, FM, and FT levels. This study indicates that IMT-CCA is negatively associated with FT levels, independent of age, total body fat, central fat accumulation, and fasting glucose concentrations in overweight and obese NGT patients. CONCLUSION: Hypotestosteronemia may accelerate the development of atherosclerosis and increase the risk for CHD in obese men. International Journal of Obesity (2003) 27, 803–807. doi:10.1038/sj.ijo.0802292 Keywords: testosterone; intima-media thickness; cardiovascular risk; atherosclerosis

Introduction Low levels of testosterone are expected in men with high body mass index (BMI) because of increased conversion of androstenedione to estrogen by the enzyme aromatase in adipose tissue1 and inhibition of the hypothalamic–pituitary–gonadal axis by leptin.2 On the other hand, low testosterone levels are responsible for higher body fat; in fact, testosterone, acting through a specific receptor,3 has lipolytic effects in adipocytes;4,5 moreover, in vivo studies

*Correspondence: Prof G De Pergola, Internal Medicine, Endocrinology, and Metabolic Diseases, Department of Emergency and Organ Transplantation, University of Bari, Piazza Giulio Cesare 11, 70124 Bari, Italy. E-mail: [email protected] Received 26 September 2002; revised 2 January 2003; accepted 26 January 2003

performed in men have shown that testosterone supplementation causes a more rapid turnover of triglycerides in the subcutaneous abdominal adipose tissue region6 and inhibits lipoprotein-lipase (LPL) activity and triglyceride uptake and assimilation,6,7 these effects being prominent in the intraabdominal fat depot.7 An inverse association has been found between plasma testosterone levels and the extent of coronary artery disease (CAD) in men with angina pectoris, thus suggesting that low testosterone levels may be a risk factor for CAD.8 The increased intima-media thickness of the common carotid artery (IMT-CCA), as measured by high-resolution B-mode ultrasound imaging, is a very early phase of the atherosclerotic process and precedes the development of plaque and stenosis in the arterial wall.9,10. IMT-CCA was found to be associated with a higher prevalence of

Free testosterone and carotid atherosclerosis G De Pergola et al

804 symptomatic and asymptomatic CAD11,12 and to correlate with future development of myocardial infarction and stroke,13 thus suggesting that the thickening of the intimamedia complex of the common carotid artery may represent a marker of coronary atherosclerosis.14,15 In particular, prospective studies have shown that the risk of acute myocardial infarction rises by 11% if an increase of 0.1 mm in the IMT-CCA occurs.16 Whether free testosterone (FT) levels in obese men are correlated with the IMT-CCA has not been investigated yet. Therefore, we studied glucose-tolerant (NGT) men with various degrees of adiposity (i) to test whether free testosterone levels are associated with an increase in IMTCCA and (ii) to evaluate whether this relation is independent of anthropometric and metabolic parameters.

Subjects and methods A total of 127 NGT overweight and obese men (BMI Z25 kg/ m2), aged 18–45 y, were enrolled for the study. NGT was defined according to the recommendations of the American Diabetes Association (ADA) Expert Committee on the Classification and Diagnosis of Diabetes Mellitus.17 Study subjects were recruited consecutively at the Outpatient Clinic of the Section on Internal Medicine, Endocrinology, and Metabolic Diseases, Department of Emergency and Organ Transplantation, University of Bari School of Medicine, where they referred to for the management of obesity. All individuals gave written informed consent to be included in the study, which was performed in accordance with the guidelines proposed in the Declaration of Helsinki. To avoid confounding factors known to affect endothelial function, coagulative pathways, and testosterone levels, the following subjects were not included in the study: smokers during the previous 12 months, subjects with diagnosed cardiovascular disease (CAD, arrythmia, and heart failure), subjects with cerebrovascular disease (stroke and transient ischemic attack), subjects with peripheral vascular disease (claudicatio intermittens and/or absence of peripheral pulses), subjects with familial and/or severe dyslipidemia (triglycerides and/or total cholesterol 4300 mg/dl), subjects with stable hypertension treated by drugs, subjects with chronic hepatic, renal, or any other serious chronic disease, and individuals both with primary or secondary hypogonadism and hyperprolactinemia. Subjects were also excluded if they were on any kind of medications. Finally, during the testing period, all subjects were asked to keep their normal mixed diet and not to perform any sporting activity. Subjects were evaluated after an overnight fast and a 24-h period of abstinence from alcohol and vigorous physical exercise. A standard 75-g OGTT was performed in each subject enrolled in the study for evaluating his glucose status and basal and 2-h postchallenge glucose concentrations. The systolic and diastolic blood pressure readings were recorded to the nearest 2 mmHg as the mean of two measurements with the subjects seated and using a mercury manometer International Journal of Obesity

with an appropriate cuff size. Central fat accumulation was evaluated by measuring the waist circumference, defined as the minimum circumference between the xyphoid process and the umbilicus. Blood samples were drawn from an antecubital vein with a 19-gauge needle. Plasma concentrations of insulin were measured by radioimmunological assay, using a commercially available kit (Behring, Scoppitto, Italy). Blood glucose was determined by the glucose-oxidase method (Sclavo, Siena, Italy). Total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglycerides were measured using enzymatic assays (Boehringer Mannheim, GMbH Diagnostica Mannheim, Germany). Insulin resistance was assessed by using the homeostatic model assessment (HOMA), based on a mathematical correlation between fasting plasma glucose and insulin levels.18 FT levels were measured on a single, pooled sample (resulting from three samples 15 min apart) by radioimmunological methods using commercially available kits, as previously described.19 Both intra- and interassay coefficients of variation were o7.5%. Body composition was estimated (fasting) by the bioimpedance method using a tetrapolar device (BIA 101/S, RJL Systems, Detroit, USA; Akern s.r.l., Florence, Italy), as previously described.20 The fat-free mass (FFM) was calculated by Heitmann’s equation21 and fat mass (FM) was calculated as the difference between body weight and FFM. IMT-CCA was determined by high-resolution B-mode ultrasound (ATL UM8; Advanced Technology Laboratories, Bothel, WA, USA), as previously described.22 All measurements were carried out by a single specialist (MC) who was blind to all clinical and laboratory characteristics. Briefly, the scanning protocol included imaging of the common and internal carotid arteries in multiple longitudinal and transverse planes. The electrocardiographic signal (lead II) was simultaneously recorded to synchronize the image capture of the top of the R-wave to minimize variability during the cardiac cycle. The near and far walls of each of four welldefined imaging sites of both carotid arteries were explored: proximal common carotid artery, 15–30 mm proximal to the carotid bulb; distal common carotid artery, o15 mm distal to the carotid bulb; proximal internal carotid artery, carotid bulb and the initial 10 mm of the vessel; and distal carotid artery, 410 mm above the flow divider. Values applied are averages of the IMT in the right and left common carotid arteries. IMT was defined as the distance from the leading edge of the lumen–intima interface to the leading edge of the media–adventitia interface of the far wall. At the position of the thickest part of the wall (visually judged), a frozen longitudinal image was captured and recorded on videotape. The procedure was repeated four times to achieve four separate images for analysis. A short sequence of real-time images was also recorded on videotape to assist in the interpretation of the frozen images. Reproducibility of the ultrasound outcome categories was nearly perfect, as

Free testosterone and carotid atherosclerosis G De Pergola et al

805 indicated by (weighted) k coefficients 40.8 for agreement between double measurements (n ¼ 100). Statistical analyses were performed using the STATISTICAs 6.0 for Windows, StatSoft Inc. (1995) software (Tulsa, OK, USA). Results are presented as average and standard error of mean (s.e.m.) for all parameters. The K–S test and the Lilliefors test for normality were performed to check whether the wide range of BMI in the study group, including both overweight (BMI 25.0–29.9 kg/m2) and obese (BMIZ30.0 kg/ m2) subjects, could be a sign of bimodal distribution. Since both tests were not significant (P40.2), it could be argued that the two subgroups, that is, overweight and obese individuals, could be tested as a whole (data not shown). Pearson’s correlation coefficients were used to quantify the univariate associations of IMT-CCA with the other variables investigated, and a multiple regression analysis was carried out to test the joint effect of different variables on IMT-CCA (dependent variable), with FT levels, age, BMI or waist circumference or FM, systolic blood pressure levels, and fasting concentrations of glucose and lipids, as independent variables. Variables with a skewed distribution (plasma levels of triglycerides and insulin) were logarithmically transformed before analysis to improve the approximation to a Gaussian distribution. The minimal statistical significance was defined for Po0.05.

Results General, anthropometric, biochemical, and ultrasonographic data of study subjects are presented in Table 1. The normal range of FT concentrations in the age range of our study subjects was 8.8–27.0 pg/ml; only eight out of 127 individuals had FT concentrations lower than the above reference range. Similarly, the normal value of IMT-CCA is r1 mm; 15.7% of study subjects had an IMT-CCA higher than the upper limit of the reference range (data not shown).

Table 1 Intima-media thickness of the common carotid artery (IMT-CCA), free testosterone levels, and other characteristics of study subjects (n ¼ 127) Variable Age (y) Body mass index (kg/m2) Fat mass (kg) Waist circumference (cm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) Fasting glucose (mmol/l) 2-h postchallenge glucose (mmol/l) Insulin (pmol/l) HOMAIR IMT-CCA (mm) Free testosterone (pg/ml)

The correlation coefficients of IMT-CCA and FT levels with the other variables are shown in Table 2. IMT-CCA was positively correlated with age (Po0.05), BMI (Po0.01), FM (Po0.01), waist circumference (Po0.05), and fasting glucose concentrations (Po0.05), and inversely associated with FT levels (Po0.001; Figure 1). FT levels were inversely associated with BMI (Po0.01), FM (Po0.01), and waist circumference (Po0.001). When IMT-CCA was considered as the dependent variable in a multiple regression analysis (fitted model: adjusted R2 0.241, F 13.114, Po0.001) with age, BMI or FM, plasma lipids (total cholesterol, HDL-cholesterol, and logarithmically transformed triglycerides), systolic blood pressure, and fasting glucose concentrations as independent variables, IMT-CCA maintained an independent association with BMI (Po0.05), FM (Po0.05), and FT levels (Po0.05). FT concentrations explained 33% of the variability of IMT-CCA.

Table 2 Pearson’s correlations between the intima-media thickness of the common carotid artery (IMT-CCA) and other anthropometric and biochemical parameters in the study subjects (n ¼ 127) IMT-CCA (mm) Age (y) Body mass index (Kg/m2) Fat mass (kg) Waist circumference (cm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) log triglycerides (mmol/l) Fasting glucose (mmol/l) 2-h postchallenge glucose (mmol/l) log insulin (pmol/l) HOMAIR Free testosterone (pg/ml)

0.23* 0.26** 0.27** 0.21* 0.09 0.09 0.06 0.14 0.05 0.22* 0.01 0.05 0.06 0.29***

Free testosterone (pg/ml)

0.25** 0.27** 0.29***

*Po0.05; **Po0.01; ***Po0.001.

Mean7s.e. 34.470.88 34.670.68 39.071.83 112.271.66 126.371.01 80.470.89 4.8370.09 1.1470.04 1.5470.06 4.7270.06 6.0970.12 183.479.72 5.3770.29 0.9170.02 17.070.55

Figure 1

Pearson’s correlation between free testosterone levels and the common carotid artery (IMT-CCA) in the study subjects (n ¼ 127).

International Journal of Obesity

Free testosterone and carotid atherosclerosis G De Pergola et al

806 When waist circumference was entered into the regression model after excluding BMI and FM (fitted model: adjusted R2 0.203, F 4.613, Po0.001), IMT-CCA maintained an independent association only with FT levels (Po0.05).

Discussion We now show that FT levels are negatively associated with IMT-CCA, independent of body fatness and other wellknown cardiovascular risk factors, thus suggesting that testosterone may have a direct antiatherosclerotic effect. Furthermore, we reported inverse associations of FT levels with BMI, FM, and waist, which are consistent with previous studies demonstrating that low plasma levels of FT are observed in obesity, particularly in abdominal/truncal obesity.23 The mechanisms responsible for the antiatherosclerotic effect of testosterone are multiple. Noteworthy, in men with CAD, i.m. testosterone treatment has been shown to have a beneficial effect on angina pectoris24,25 and exercise-induced ST-segment depression26,27 and to enhance endotheliumindependent coronary artery dilation and flow-mediated brachial arterial vasoreactivity.28,29 A more recent study has shown that even long-term oral administration of testosterone enhances endothelium-dependent and endotheliumindependent vasodilation.30 Moreover, experiments performed in castrated male animal models have shown that supplements of androgens inhibit atheroma formation in animals23 and that testosterone is primarily inhibiting the calcium-dependent elements of vascular contraction.30 In addition, it has been shown that testosterone relaxes porcine coronary arteries by opening the largeconductance, calcium-activated potassium channel,31 and that it attenuates expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), thus inhibiting the adhesion of monocytes to endothelial cells.32 This phenomenon may help explain the mechanism by which testosterone may have beneficial effects on the cardiovascular system; however, this effect is mediated by endothelial cell aromatase, converting testosterone to estradiol.32 IMT-CCA has been shown to be associated with the prevalence of cardiovascular disease33 and atherosclerotic involvement of other arterial beds, including the coronary arteries,11,12 and to represent a strong predictor of both myocardial infarction and stroke,13 even in subjects with no history of cardiovascular disease.9 Therefore, the results from this study based on IMT-CCA measurements in overweight and obese men may be potentially extrapolated to suggest that hypotesteronemia enhances the susceptibility to atherosclerosis in multiple arterial districts and increases the risk for CHD. The independent association between low testosterone levels and IMT-CCA in young healthy adults points out to the fact that these subjects may be considered to be already affected by an early stage of atherosclerosis. International Journal of Obesity

IMT-CCA was positively correlated with age, BMI, FM, waist circumference, and fasting glucose concentrations, thus confirming the results of our previous studies.34,35 However, after multivariate analysis, IMT-CCA maintained an independent association only with FM (or BMI) and FT, thus definitely showing that body fatness per se has a deleterious effect on the development of the atherosclerotic process. At variance with our previous findings,36 insulin resistance was not associated with the thickness of the arterial wall. However, it is noteworthy that insulin sensitivity was estimated by HOMAIR in the present study and directly measured in our previous study showing an independent relation between insulin resistance and the IMT-CCA.36 Even though blood pressure levels and lipids are wellknown cardiovascular risk factors, these parameters did not show a significant relation with the IMT-CCA. However, it has to be pointed out that subjects enrolled in this study had neither severe dyslipidemia nor arterial hypertension treated by drugs. In conclusion, the present study, mostly performed in obese men, demonstrates that low testosterone levels are associated with higher thickness of the common carotid artery. Since the thickening of the intima-media complex in the carotid artery may be an early asymptomatic sign of atherosclerosis and coexists with similar changes in the coronary arteries, this study indicates that hypotestosteronemia may accelerate the development of atherosclerosis and increase the risk for CHD in obese patients.

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International Journal of Obesity