Endothelium-dependent flow-mediated vasodilatation, insulin resistance and the metabolic

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1 Journal of Internal Medicine 2002; 252: Endothelium-dependent flow-mediated vasodilatation, insulin resistance and the metabolic syndrome in 60-year-old men I. WENDELHAG 1, B. FAGERBERG 2, J. HULTHE 1, L. BOKEMARK 2 & J. WIKSTRAND 1 From the 1 Wallenberg Laboratory for Cardiovascular Research; and 2 Institute of Internal Medicine, Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden Abstract. Wendelhag I, Fagerberg B, Hulthe J, Bokemark L, Wikstrand J (Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden). Endothelium-dependent flow-mediated vasodilatation, insulin resistance and the metabolic syndrome in 60-year-old men. Journal of Internal Medicine 2002; 252: Objectives. To evaluate the endothelium-dependent flow-mediated vasodilatation (FMD) in the brachial artery and to study the relationship to insulin sensitivity and to the metabolic syndrome in 60- year-old clinically healthy men. Subjects. The men were randomly selected from the general population (n ¼ 55). The subjects with the metabolic syndrome were defined according to a definition proposed by a working group associated with the World Health Organization (WHO). Methods. Ultrasound images for measurement of lumen diameter of the brachial artery were recorded before and after reactive hyperaemia induced by occlusion of the artery, both with and without ischaemic hand exercise during the occlusion. Insulin-mediated glucose uptake was determined by euglycaemic hyperinsulinaemic clamp as a measure of insulin sensitivity. Results. The FMD was in the total group 3.2% when hyperaemia was induced by occlusion only and 8.7% after occlusion plus ischaemic hand exercise (P < 0.001, n ¼ 51). However, no relationship was observed between any measure of FMD and insulin-mediated glucose uptake (r ¼ )0.05 and r ¼ 0.06, n ¼ 47, P > 0.30). Furthermore, subjects with the metabolic syndrome (n ¼ 13) did not differ in any measure of FMD compared with those with no risk factors (n ¼ 11). Conclusion. In this study the ultrasound method to evaluate endothelial function did not show that low insulin sensitivity or the metabolic syndrome were associated with impaired FMD in otherwise clinically healthy 60-year-old men. Keywords: brachial artery, endothelial function, hyperaemia, ultrasound. Introduction The normal endothelium contributes to local regulation of vasomotor tone, prevents thrombosis, controls transport of solutes and macromolecules, inhibits oxidative processes, regulates the adhesion of leucocytes, and participates in the control of smooth muscle cell proliferation [1]. Several of these mechanisms are regulated by nitric oxide (NO) that is produced by the endothelium. The arterial endothelium is a target for the atherosclerotic process and it has been shown that atherosclerotic disease is associated with endothelial dysfunction, and this can be demonstrated very early in the disease process [2]. Ageing and exposure to risk factors such as hypercholesterolaemia, hypertension and smoking are also associated with endothelial dysfunction [3 6]. Of other potential factors, insulin resistance is of particular interest. Several of the cardiovascular risk factors that are associated with insulin resistance, constituting the metabolic or insulin resistance syndrome [7, 8] may be accompanied by endothelial dysfunction [9 11]. In addition, the vascular Ó 2002 Blackwell Science Ltd 305

2 306 I. WENDELHAG et al. and metabolic action of insulin is partly mediated by NO and it has been suggested that impaired NO synthesis may be a central defect underlying the metabolic syndrome and the increased risk of atherosclerotic disease [12]. Flow-mediated vasodilatation (FMD) of the brachial artery is a measure of endothelial-dependent, NO-regulated function [3 5, 13, 14]. The weakness of this method is that the postocclusion vasodilatation response is very small above the age of 60 years, i.e. an age when atherosclerotic diseases are getting more prevalent [4]. However, in previous methodological studies we observed that FMD could be increased significantly after adding ischaemic hand exercise during occlusion and that this increase was NO-dependent [15, 16]. The aim of this study was to evaluate the endothelial-dependent vasodilatation in the brachial artery and to study the relationship to insulin sensitivity and to the metabolic syndrome in 60- year-old clinically healthy men. Methods Study group This investigation was part of the Atherosclerosis and Insulin Resistance (AIR) Study [17]. The inclusion criteria in this study were age 58 years at enrolment, male sex and Swedish ancestry. Exclusion criteria included cardiovascular disease defined as a history of myocardial infarction, angina pectoris, heart failure, stroke, intermittent claudiocatio and treatment for hypertension or hyperlipidaemia. Other established disease was symptomatic chronic disease such as underlying rheumatoid arthritis, alcoholism, malignancy and psychiatric disorders. No subjects with clinically overt diabetes were included. The patients were randomly selected amongst men in the County Council register and were invited to a screening examination. The screening protocol aimed at enriching the population sample with men with low and high insulin sensitivity, respectively. Thus, in connection with the screening examination the subjects were divided into quintiles of a body mass index (BMI)/ blood glucose score, which allowed immediate stratification and selection for further studies. This has previously been described in detail [17]. A random sample of every fourth man in quintile 1 (indicating low insulin sensitivity) and quintile 5 (indicating high sensitivity) and every fifth man in quintile 2 4 (indicating intermediate sensitivity) were invited to further examinations. A total of 818 men performed the screening examination and from this group of men 391 subjects were selected for future cross-sectional and prospective studies of insulin metabolism and ultrasound examination of the carotid and femoral arteries. In 104 randomly selected men from this group a euglycaemic hyperinsulinaemic clamp examination was performed. A further sub sample of 55 men from quintiles 1 and 5 was selected for ultrasound examination of the brachial artery for evaluation of endothelial function. Good quality images from the brachial artery were missing in four subjects. Forty-seven subjects in this group had undergone euglycaemic hyperinsulinaemic clamp examination. All men were 60- years old when the examinations of the brachial arteries were performed. All subjects gave informed consent to participate, and the study was approved by the local Ethics Committee. Definition of the metabolic syndrome A definition of the metabolic syndrome has been suggested by a working group consulted by World Health Organization (WHO) in 1998 [8]. According to this definition the metabolic syndrome is defined as insulin resistance together with two or more of the following risk factors: (i) Raised arterial (systolic/ diastolic) pressure (7160/90 mmhg, either value); (ii) Raised triglycerides (71.7 mmol L )1 ) and/or decreased high-density lipoprotein (HDL) cholesterol (<0.9 mmol L )1 ); (iii) Central obesity (waist to hip ratio >0.90 and/or BMI >30 kg m )2 ); and (iv) microalbuminuria (urinary albumin excretion rate 720 lg min )1 or albumin : creatinine ratio 720 mg g )1 ). Thirteen subjects fulfilled the criteria for the metabolic syndrome. Twenty-three subjects had at least one of the above risk factors and 11 patients had no risk factors at all. Biochemical analysis Serum concentrations of total cholesterol and triglycerides were determined by fully enzymatic techniques [18, 19]. HDL was determined after precipitation of apolipoprotein B-containing lipoproteins

3 ENDOTHELIAL FUNCTION AND METABOLIC SYNDROME 307 with Mn-chloride and dextran sulphate. Low-density lipoprotein (LDL) was calculated as described by Friedewald et al. [20]. Whole glucose was measured with the glucose oxidase technique. Total plasma insulin was determined in all subjects with a radioimmunoassay (Pharmacia Insulin RIA, Pharmacia Diagnostics, Sweden). Intact proinsulin and 32, 33 split proinsulin were assayed in duplicate using a time resolved fluorometric assay (DELFIA). The solid phase antibody, bound to a microtitre plate was the same in each case (Sobey, 1989). The labelled antibody used in the 32, 33 split proinsulin assay (CPT 3f11) was produced by Daho Diagnostics (Copenhagen, Denmark). The intact proinsulin assay shows <1% cross-reaction with insulin and 32, 33 split proinsulin at concentrations of 2500 and 400 pmol L )1, respectively. Between batch coefficients of variation are 8.5% at 20 pmol L )1. LDL cholesterol peak particle size was determined by gradient gel electrophoresis as described earlier [21]. Ultrasound examination of the brachial artery B-mode ultrasound was used to record longitudinal images of the right brachial artery 5 10 cm above the elbow. The ultrasound scanner was an Acuson 128 with a 7-MHz-EF linear transducer. The examinations were performed in a temperature-controlled room (24 C). Electrocardiographic signal (lead II) was simultaneously recorded to synchronize the image capture to the top of the R wave in order to minimize variability during the cardiac cycle. The subject s right arm was comfortably immobilized in the extended position by help of a vacuum pillow and the transducer mounted in a stereotactic clamp in order to keep the same position throughout the examination. The transducer position was also marked on the skin with a marker pen, which helped when small adjustments of the transducer were needed [15]. After 10 min rest, ischaemia was induced by inflation of a pneumatic tourniquet around the forearm with a pressure 50 mmhg above systolic blood pressure during 5 min. The cuff was placed distal to the area where lumen diameter was measured, which means that this area was never ischaemic. Images for measurement of lumen diameter were recorded before and during 2 min after cuff pressure release. Blood flow was intermittently recorded using Doppler technique. After 10 min and a new resting scan ischaemia was again induced by a pressure 50 mmhg above systolic blood pressure, but after 1 min of occlusion, the subject was also instructed to pump a rubber bladder. The ischaemic hand exercise continued as long as the subject was able to pump the rubber bladder in order to reach maximal ischaemia (approximately min depending on physical capacity). The cuff pressure was then released and images for measurement of lumen diameter were recorded during 3 min after pressure release [15]. After a new period of 10 min rest 0.5 mg nitroglycerine was administered sublingually, giving a vasodilatation of the artery by a mechanism that is independent of the endothelium. Images were recorded before and 4 min after administration. Analysis of ultrasound images The ultrasound images from the videotapes were analysed in a computerized analysing system for automated measurements [22]. The measurements of lumen diameter were made along a 10-mm-long segment in the brachial artery and defined by the distance between the leading edge of the intima lumen interface of the near wall and the lumen intima interface of the far wall. A correction of the automated outlining of the interfaces was only performed when an obvious error occurred. Lumen diameter was measured approximately every 20 s during the recording after the pressure release. The measurement results were not displayed on the monitor during analysis to avoid bias. Flow-mediated vasodilatation was defined as the maximal increase in lumen diameter after cuff pressure release expressed as a percentage change of lumen diameter at rest. Blood flow velocity was measured from the Doppler recordings. Assessment of insulin sensitivity by euglycaemic hyperinsulinaemic clamp Before the examination, the subjects were asked not to change any habits. During the 2 days preceding the day of examination, subjects were to avoid unusual physical exercise, alcohol consumption or any major change in caloric intake. Subjects had to fast and avoid medication, smoking or snuff-taking

4 308 I. WENDELHAG et al. from midnight the preceding day; subjects were allowed to drink water in the morning on the day of examination. Before the examination started, a questionnaire was completed to verify that the subject had followed the instructions and that there were no signs of respiratory infection or fever. The euglycaemic hyperinsulinaemic clamp examination was performed ad modum DeFronzo et al. [23], slightly modified according to a previous description [24]. After a priming dose, the insulin infusion rate was 1 ml min )1 kg )1 body weight, continuing for 120 min until the end of the examination. During the clamp, the target whole blood glucose concentration was 5 mmol L )1 and the glucose infusion rate was adjusted in connection with each determination of whole blood glucose if necessary. After the clamp examination, fat-free mass was measured using the dual-energy X-ray absorptiometry body composition model (Lunar DPX-L, Madison, WI, USA). Insulin sensitivity was calculated as the glucose infusion rate per minute adjusted for fat-free mass (GIR FFM ) during the final hour of examination. Statistics All statistics were analysed using SPSS for Windows 9.0. The Mann Whitney U-test was used for comparison between groups. Wilcoxon s paired test was used for comparison between the two methods to induce a flow increase, with and without ischaemic hand exercise. A t-distributed variable was used to calculate 95% confidence intervals (CIs) for differences. Nonparametric Spearman s rank correlation test was used in the correlation analysis with the relationship illustrated with Pearson s correlation coefficient (r). P-values less than 0.05 (two-sided) were regarded as statistically significant. Results Characteristics of the subjects with the metabolic syndrome Subjects with the metabolic syndrome had as expected higher blood pressure, BMI, serum triglycerides, lower HDL levels and also higher glucose and insulin values compared with subjects with no risk factors (not tested for statistical significance because of selection criteria, Table 1). There were no differences in heart rate (HR), total cholesterol, LDL cholesterol or smoking habits between the groups. However, a smaller LDL peak particle size was seen in the subjects with the metabolic syndrome compared with the subjects with no risk factors (P < 0.001, Table 1). Table 1 Characteristics of the study participants Variables I. Subjects with no risk factors (n ¼ 11) II. Subjects with one or more risk factors (n ¼ 23) III. Subjects with the metabolic syndrome (n ¼ 13) Difference between I and III 95% CI for differences P-value SBP (mmhg) 121 ± ± ± ()4 25) NA DBP (mmhg) 74 ± 6 77 ± ± 8 7 (1 13) NA HR (beats per minute) 58 ± 8 59 ± 8 60 ± 3 2 ) BMI (kg m )2 ) 21.9 ± ± ± (8 13) NA Present smokers (%) )3 >0.30 Past smokers (%) Total cholesterol (mmol L )1 ) 5.77 ± ± ± 1.04 )0.07 ) >0.30 LDL cholesterol (mmol L )1 ) 3.91 ± ± ± 0.80 )0.06 ) >0.30 HDL cholesterol (mmol L )1 ) 1.45 ± ± ± 0.17 )0.41 ()0.64 )0.18) NA Serum triglycerides (mmol L )1 ) 0.90 ± ± ± ( ) NA Blood glucose (mmol L )1 ) 4.4 ± ± ± NA Plasma insulin (pmol L )1 ) 35.1 ± ± ± NA Plasma proinsulin (pmol L )1 ) 7.7 ± ± ± NA Plasma 32, 33 split 8.7 ± ± ± NA proinsulin (pmol L )1 ) LDL particle size (nm) 26.9 ± ± ± 0.7 )1.1 )1.6 ))0.6 <0.001 SD, standard deviation; CI, confidence interval; SBP/DBP, systolic and diastolic blood pressure; LDL, low density lipoprotein; HDL, high density lipoprotein; NA, not applicable as these variables are selection criteria.

5 ENDOTHELIAL FUNCTION AND METABOLIC SYNDROME 309 FMD and insulin sensitivity Blood flow velocity was significantly higher after ischaemic hand exercise during occlusion, 1.50 m s )1, compared with after occlusion only, 1.21 m s )1 (n ¼ 51, P < 0.001) in the total group. Also FMD was significantly higher when hyperaemia was induced by occlusion plus ischaemic hand exercise, 8.7%, compared with 3.2% after occlusion only (n ¼ 51, P < 0.001). All subjects responded with an arterial dilatation after the administration of nitroglycerine. Insulin-mediated glucose uptake adjusted for fat free mass, as a measure of insulin sensitivity, was 4.1 ± 1.2 mg kg )1 min )1 in the patients with the metabolic syndrome (n ¼ 13) compared with 10.6 ± 2.3 mg kg )1 min )1 in the patients with no risk factors (n ¼ 11, P < 0.001). No relationship was observed between FMD and insulin-mediated glucose uptake, neither after occlusion only nor after occlusion plus ischaemic hand exercise in the total group (r ¼ )0.05 and r ¼ 0.06, n ¼ 47, P > 0.30). Neither were there any relationships between FMD and s-insulin (r ¼ 0.07 and r ¼ )0.05, n ¼ 50, P > 0.30) nor FMD and pro-insulin (r ¼ 0.02 and r ¼ )0.13, n ¼ 50, P > 0.30). FMD and the metabolic syndrome There were no differences in lumen diameter or FMD between the subjects with the metabolic syndrome and the subjects with no risk factors (Table 2). FMD after occlusion plus ischaemic hand exercise was 8.4 ± 3.0% in the group with the metabolic syndrome compared with 10.1 ± 5.9% in the group with no risk factors (P > 0.30, Table 2). The percentage increase in lumen diameter during the time after reactive hyperaemia induced by occlusion only and also by occlusion plus ischaemic hand exercise is illustrated in Fig. 1. Discussion The results from this study did not show any association between low insulin sensitivity and an impaired endothelium-dependent vasodilatation. Furthermore, the subjects with the metabolic syndrome did not significantly differ in FMD from the subjects who had no risk factors in this syndrome. To our knowledge there were no published data on FMD in subjects with the metabolic syndrome. There are, however, several studies of the relation between insulin sensitivity and endothelial function. Only one of these studies measured FMD in the brachial artery by using the ultrasound method [25]. In that study, normotensive, normoglycaemic first-degree relatives to patients with type 2 diabetes mellitus were compared with normal controls. Insulin resistance was associated with impaired FMD, also in a multiple regression analysis. In comparison with our study, their subjects were more than 20 years younger with an FMD that Table 2 Blood flow velocity and lumen diameter of the brachial artery recorded before and after a reactive hyperaemia Variables I. Subjects with no risk factors (n ¼ 11) II. Subjects with one or more risk factors (n ¼ 23) III. Subjects with the metabolic syndrome (n ¼ 13) Difference between I and III 95% CI for differences P-value Hyperaemia, occlusion only Resting diameter (mm) 4.11 ± ± ± ) Maximal diameter (mm) 4.20 ± ± ± ) >0.30 FMD (%) 2.45 ± ± ± ) >0.30 Resting flow (m s )1 ) 0.68 ± ± ± ) >0.30 Maximal flow (m s )1 ) 1.17 ± ± ± ) Hyperaemia, occlusion + ischaemic hand exercise Resting diameter (mm) 4.04 ± ± ± ) Maximal diameter (mm) 4.44 ± ± ± ) >0.30 FMD (%) ± ± ± 3.00 )1.75 ) >0.30 Resting flow (m s )1 ) 0.71 ± ± ± ) >0.30 Maximal flow (m s )1 ) 1.53 ± ± ± 0.27 )0.03 ) >0.30 SD, standard deviation; CI, confidence interval; FMD, flow-mediated vasodilatation.

6 310 I. WENDELHAG et al. Change in lumen diameter (%) W Occlusion only Occlusion + ischaemic exercise Time (s) I. No risk factors (n = 11) II. One risk factor (n = 23) III. Metabolic syndrome (n = 13) Fig. 1 Percentage increase in lumen diameter in three different groups during 120 s after reactive hyperaemia induced by 5 min brachial artery occlusion (left), and during 180 s after reactive hyperaemia induced by 1 min brachial artery occlusion plus 1 min ischaemic hand exercise during occlusion (right). was two to three times higher in the insulinsensitive group. The remaining seven studies can be divided into five studies that also measured insulin sensitivity by using the euglycaemic hyperinsulinaemic clamp method [26 30] and two studies where indirect methods were used [31, 32]. In the first group of five studies, endothelial function was assessed by plethysmography as blood flow response in the forearm [26, 28 30] or the leg [27] to intra-arterial infusion of acetylcholine or metacholine. One of these five studies was positive [27], demonstrating that acetylcholine-induced increase in blood flow in the leg was correlated with insulin sensitivity whereas four showed no such associations [26, 28 30]. In the study by Petrie et al. there was an association between insulin-mediated glucose uptake and the response to L-NMMA (NG-monomethyl-L-arginine) [26]. One of the negative studies also included a randomized controlled substudy with troglitazone, an insulin-sensitizer, demonstrating that this compound, on one hand increased insulin-induced forearm glucose uptake, but, on the other hand, did not affect endothelial function [30]. In one of the two remaining studies insulin sensitivity was measured with the frequently sampled intravenous glucose tolerance test in diabetic patients and healthy controls [31]. The forearm blood flow response to intra-arterial acetylcholine was found to correlate with insulin sensitivity. In another of the previously published studies, insulin sensitivity was estimated with plasma insulin concentration and homeostasis model assessment (HOMA) [32]. In this study patients without angiographically significant atherosclerotic disease were examined with an acetylcholine provocation test in the left and right coronary arteries. Those who showed a pathological response, indicating

7 ENDOTHELIAL FUNCTION AND METABOLIC SYNDROME 311 endothelial dysfunction, had higher plasma insulin and lower insulin sensitivity as evaluated by HOMA level compared with the remaining patients. There were no differences in other risk factor levels between the groups. When comparing the results of the present and previous studies a number of methodological issues have to be addressed. Firstly, the characteristics of the individuals who were examined varied between the studies. Five studies, including the present one, were negative and did not observe any association between insulin sensitivity and endothelial function measured as FMD or the response to acetylcholine or methylcholine [26, 28 30]. Amongst these, three studies investigated healthy individuals with varying degrees of overweight [26, 28, 30], one study encompassed hypertensive men [29], whilst the present study included clinically healthy men with and without the metabolic syndrome. The four positive studies included patients with diabetes mellitus [25, 27, 31] or severe obesity [27], firstdegree relatives to diabetics [25], healthy controls [25, 27, 31] and patients suspected of having ischaemic heart disease [32]. Hence, positive findings have only been obtained amongst patients with diabetes mellitus or heredity for this disease or patients suspected to suffer from ischaemic heart disease and not amongst clinically healthy individuals. The importance of diabetes is further indicated by a study of 44 patients with type 2 diabetics, but without any of the other cardiovascular risk factors constituting the metabolic syndrome. The patients assigned to metformin treatment improved insulin resistance, estimated by the homeostasis model, and endothelial function assessed by blood flow response to intra-arterial acetylcholine and there was a strong statistical link between these variables [33]. Secondly, age is of importance as endothelialdependent vasodilatation decreases in parallel with increasing age, either this is measured as blood flow response to intra-arterial acetylcholine or as FMD [4, 34]. Paradoxically, some of the negative studies had the youngest subjects with a mean age below 40 years [28, 30] whereas the age ranged from 35 to 59 years in the positive studies [27, 31, 32]. It is obvious that age is a powerful confounder and that it is important to control for this factor. The present study was the only one that kept the age factor constant by only recruiting men of the same age. The age in the present study, 60 years, was chosen in order to include subjects with a high risk of atherosclerotic disease. Thirdly, the results from the available studies may have been affected by type I or II errors because of inadequate sample sizes. However, the present study is the second largest compared with the published studies above. Fourthly, methodological differences may also have influenced the results. With plethysmography it is possible to assess changes in blood flow which, however, may be caused by several mechanisms. Thus, metacholine has several potential mechanisms of action that could reduce vascular resistance including receptor-stimulated release of NO, activation of vasodilator prostanoids and inhibition of noradrenaline release from sympathetic nerve endings [9]. Ultrasound assessment of brachial artery diameter allows a direct measurement of vasodilatation. The drawback is that the postocclusion response is very small in subjects above the age of 60 years [4]. Adding ischaemic hand exercise may be of value when quantifying endothelial dysfunction in this age group [16]. The method to increase vasodilatation by adding ischaemic hand exercise has been validated by us by measuring lumen diameter at rest and after reactive hyperaemia induced by occlusion with and without hand exercise during NaCl or L-NMMA infusion in healthy 60- year-old men. The percentage increase in postocclusion lumen diameter was significantly lower after ischaemic hand exercise with intra-arterial infusion of L-NMMA compared with infusion of NaCl (P < 0.05). This indicates that the increased postocclusion vasodilatation of the brachial artery after ischaemic hand exercise is NO mediated [16]. Taken together, we believe that we have used a method that increases the possibilities to observe changes in FMD in higher ages also. However, as in most other studies we examined FMD with the subjects in a fasted state, and it cannot be excluded that this may have diminished a possible difference between the men with and without the metabolic syndrome. The metabolic syndrome is characterized by postprandial hyperlipidaemia, that is known to impair endothelium-dependent vasodilatation [35]. In conclusion, in this study, the ultrasound method to evaluate endothelial function did not show that either low insulin sensitivity or the

8 312 I. WENDELHAG et al. metabolic syndrome were associated with impaired FMD in otherwise clinically healthy 60-year-old men. Acknowledgements This study was supported by grants from the Swedish Heart-Lung Foundation, the Swedish Medical Research Council (10880), and AstraZeneca, Mölndal, Sweden. References 1 Vanhoutte PM. Endothelial dysfunction and atherosclerosis. Eur Heart J 1997; 18 (Suppl. E): E Anderson TJ, Gerhard MD, Meredith IT et al. Systemic nature of endothelial dysfunction in atherosclerosis. Am J Cardiol 1995; 75: 71B 4B. 3 Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol 1994; 24: Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging is associated with endothelial dysfunction in healthy men years before the agerelated decline in women. 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9 ENDOTHELIAL FUNCTION AND METABOLIC SYNDROME Hogikyan RV, Galecki AT, Pitt B, Halter JB, Greene DA, Supiano MA. Specific impairment of endothelium-dependent vasodilation in subjects with type 2 diabetes independent of obesity. J Clin Endocrinol Metab 1998; 83: Inoue T, Matsunaga R, Sakai Y, Yaguchi I, Takayanagi K, Morooka S. Insulin resistance affects endothelium-dependent acetylcholine-induced coronary artery response. Eur Heart J 2000; 21: Mather KJ, Verma S, Anderson TJ. Improved endothelial function with metformin in type 2 diabetes. J Am Coll Cardiol 2001; 37: Taddei S, Virdis A, Ghiadoni L, Salvetti G, Bernini G, Magagna AS. Age-related reduction of NO availability and oxidative stress. Hypertension 2001; 38: Vogel RA, Corretti MC, Plotnick GD. Effect of a single high-fat meal on endothelial function on healthy subjects. Am J Cardiol 1997; 79: Received 17 April 2000; revision received 26 July 2002; accepted 1 August 2002 Correspondence: Dr. Inger Wendelhag, Wallenberg Laboratory Fack 16, Sahlgrenska University Hospital, S Gothenburg, Sweden (fax: ; inger.wendelhag@zeta.telenordia.se).