Metformin improves endothelial function in ... - Wiley Online Library

Journal of Internal Medicine 2005; 258: 250–256

Metformin improves endothelial function in patients with metabolic syndrome C. VITALE1, G. MERCURO2, A. CORNOLDI1, M. FINI1, M. VOLTERRANI1 & G. M. C. ROSANO1 From the 1Department of Medical Sciences, Cardiovascular Research Unit, IRCCS San Raffaele–Roma, Tosinvest Sanita’, Rome; and 2Department of Cardiology, University of Cagliari, Cagliari; Italy

Abstract. Vitale C, Mercuro G, Cornoldi A, Fini M, Volterrani M, Rosano GMC (IRCCS San Raffaele– Roma, Rome; and University of Cagliari, Cagliari; Italy). Metformin improves endothelial function in patients with metabolic syndrome. J Intern Med 2005; 258: 250–256. Background. Metabolic Syndrome (MS) is associated with impaired endothelial function and increased cardiovascular risk. Insulin resistance is a key feature of MS and plays an important role in the pathogenesis of endothelial dysfunction. Aim of the present study was to evaluate the effect of metformin on endothelial function and insulin resistance, assessed by the homeostasis model (HOMA-IR, homeostasis model assessment-insulin resistance), in patients with MS. Methods. Sixty-five subjects (37 men and 28 women, mean age 54 ± 6 years) with MS were allocated to receive metformin 500 mg twice daily (n ¼ 32) or placebo (n ¼ 33) for 3 months. Before and after treatment we assessed endothelial function, using flow-mediated dilatation of the brachial artery, and HOMA-IR.

Introduction Metabolic Syndrome (MS) is a highly prevalent and complex clinical entity that associates abdominal obesity, insulin resistance or glucose intolerance, high blood pressure, hypertriglyceridaemia and low HDL cholesterol and that is associated with an increased cardiovascular risk [1–3]. Insulin resistance was suggested by Reaven as the cause of ‘metabolic Syndrome X’ and is now considered an important component of the MS 250

Results. Patients who received metformin demonstrated statistically significant improvement in endothelium-dependent vasodilation compared with those treated with placebo (from 7.4 ± 2.1% to 12.4 ± 1.9% vs. 7.3 ± 2.5% to 6.9 ± 2.7%, P ¼ 0.0016, metformin vs. placebo respectively), without significant effect on endothelium-independent response to sublingual glyceryl trinitrate (P ¼ 0.32). Metformin improved insulin resistance compared with placebo group (HOMA-IR from 3.39 to 2.5 vs. 3.42 to 3.37; 26% reduction in HOMA-IR, P ¼ 0.01). An association between the improvement in insulin resistance and the improvement in endothelial function (r ¼ )0.58, P ¼ 0.0016) was found. Conclusion. Metformin improves both endothelial function and insulin resistance in patients with MS. These findings support the central role of insulin resistance in the development of endothelial dysfunction and the role of metformin for the treatment of patients with MS. Keywords: endothelial function, insulin resistance, metabolic syndrome, metformin.

linked to both visceral obesity and arterial hypertension [4]. Insulin resistance has also been suggested as an independent risk factor of coronary heart disease as insulin-resistant subjects have a higher incidence of cardiovascular events compared with subjects with normal insulin sensitivity [3, 5–7]. Recent data suggest that patients with insulin resistance and MS have an impaired endothelial function and that this attenuation may represent an initial step towards the development of atherosclerosis, and to the increased cardiovascular risk [8–11].
2005 Blackwell Publishing Ltd

METFORMIN IN METABOLIC SYNDROME

Hyperinsulinaemia and insulin resistance are associated with an impaired NO release from endothelial cells and with reduced endothelial function. Metformin has been consistently shown to lower fasting insulin levels in type 2 diabetes [12–14] and in insulin-resistant states [15, 16]. Several studies have suggested that metformin, an insulin-sensitizing biguanide, may improve some of the features of the MS as it not only improves insulin sensitivity in the liver and muscle, as its primary anti-hyperglycaemic mechanism of action, but also induces additional beneficial effects on several metabolic abnormalities associated with the MS [16]. At the present it is not clear whether in patients with MS the improvement of insulin resistance after therapy with metformin may in turn improve endothelial function. Aim of this study was to evaluate the effect of metformin on insulin resistance and endothelial function in patients with MS.

Methods Study population The study population included 78 consecutive newly diagnosed patients with the clinical features of MS, defined according to the criteria proposed by the World Health Organization [17]. Included in the study were those 65 (37 men, 28 women, mean age 54 ± 6 years) with insulin resistance, without any change in dietary habits and physical activity, stable blood pressure values and lipid and glycaemic profile over the past 3 months, and who had not previously received any therapy for glycaemic control or for insulin resistance. Patients with contraindications to metformin therapy, including renal or hepatic impairment, congestive heart failure, severe respiratory insufficiency, acute or chronic metabolic acidosis, alcoholism, dehydration, need of use of intravenous radiographic contrast agents and those with overt diabetes mellitus and known intolerance to metformin were excluded from the study. Study design The study design was randomized, parallel, placebo controlled. After a baseline evaluation of all inclusion and exclusion criteria, all suitable patients entered a run in phase, to ensure the stability of

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clinical and laboratory parameters of MS for 4 weeks, at the end of which patients underwent a baseline evaluation of flow-mediated dilatation (FMD) of the brachial artery and of whole body insulin resistance by the homeostasis model (HOMAIR, homeostasis model assessment-insulin resistance). All patients were given a low carbohydrate normo-caloric diet and all were randomized to receive, in blind, either metformin (500 mg, b.d.s.) or matching placebo (b.d.s.) for 3 months, added to background therapy. All patients underwent re-evaluation of FMD of the brachial artery and HOMA-IR at the end of the study. The study protocol was approved by the San Raffaele Eur Ethical Committee and all subjects gave informed consent prior to participation. Patients and controls were asked to refrain from smoking and drinking alcohol or caffeine-containing beverages and, also, to abstain from severe physical exercise in the 24 h preceding the study. At each visit, patients were weighed and measured to calculate body mass index (BMI) and spent at least a 20-min acclimatization period lying in a quiet room at a controlled temperature of 20 ± 1 C and relative humidity of 65 ± 10%. Blood samples for fasting serum insulin, glucose, haemoglobin A1c (HbA1c), cholesterol (total, HDL-c, LDL-c), and triglycerides were obtained 20 min prior the investigation of brachial artery endothelial function and chemical parameters were evaluated with standard methods. Whole-body IR was calculated using the HOMA-IR [18]. Brachial artery endothelial function Endothelial function was assessed by measuring changes from baseline in the calibre of the brachial artery during reactive hyperaemia, a procedure that increases blood flow and sheer stress through the vessel. Brachial artery FMD was measured with high-resolution ultrasound. The same investigator studied each patient in order to avoid inter-observer variability. Studies of brachial artery reactivity were conducted according to a previously reported protocol [19]. In brief, all patients were studied in a quiet, temperature-controlled room (22–23 C). Participants were asked to avoid drinking beverages containing caffeine and to refrain from smoking for 6 h preceding the study. After 15-min of rest in a supine position, the right brachial artery was


2005 Blackwell Publishing Ltd Journal of Internal Medicine 258: 250–256

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imaged using a Hewlett-Packard 2500 high-resolution ultrasound machine (Hewlett-Packard, Palo Alto, CA, USA) equipped with a 7.5–12.5 MHz linear array transducer. The artery was scanned over a longitudinal section 3–5 cm above the elbow, the site where the clearest image can be obtained. The focus zone was set to the depth of the anterior vessel wall. Depth and gain settings were optimized to identify the lumen vessel wall interface. The diameter of the right brachial artery was measured continuously four times: at rest, during reactive hyperaemia, after a 10-min recovery period, and 5 min following sublingual nitroglycerin. A pneumatic tourniquet was placed around the forearm distal to the target artery and has been inflated to a pressure of 50 mmHg above patients’ systolic blood pressure for 5 min. Reactive hyperaemia was induced by sudden cuff deflation. The brachial artery was continuously imaged for 30 s prior to and 180 s after cuff release. Ten minutes following cuff deflation, a third scan was recorded to confirm that basal conditions had been re-established. To assess endothelium-independent vasodilation, sublingual nitroglycerin (0.4 mg) was administered, and a fourth scan was recorded for 5 min. The ultrasound images were recorded directly onto the hard disk of the ultrasound machine, and transferred to a floppy disk. Image analysis was performed using a validated program. The diameter of the brachial artery was measured from the anterior to the posterior interface. For each condition, the mean arterial diameter was calculated from four cardiac cycles synchronized with the R-wave peak on the electrocardiogram. All measurements were made at end diastole. The diameter change was expressed as the percentage change compared with baseline diameter. In our group the intra-observer variability in diameter measurements is 0.38 ± 0.26% (range 0.1–1.2), the coefficient of variation is 1.26% and the coefficient of repeatability is 0.5%, as previously reported. Biochemical parameters Blood samples were obtained at baseline and after 3 months. Venous blood samples were taken after at least 10 h fasting, in a supine position after 20 min of rest with a Vacutainer system (Becton Dickinson, Meylan, France). Baseline blood samples were collected in tubes containing serum to evaluate with

standard methodologies insulin, glucose, cholesterol (total, HDL-c, LDL-c), and triglycerides levels and tubes with ethylenediaminetetraacetic acid for HbA1c. The HOMA-IR was used as a measure of insulin resistance where HOMA-IR is calculated as fasting serum insulin (uU mL)1) · fasting glucose (mmol L)1)/22.5. Statistical analysis Values are given as mean ± SD or as percentages where appropriate. Analysis of covariance was performed in order to test statistical differences (treatment effect) between groups. ancova for repeated measurements using baseline values as constant covariates was used to test statistical differences between baseline and follow-up measurements between groups. Correlation between variables was calculated using the Spearman’s correlation coefficient. A stepwise multivariate analysis was performed in order to detect the variables associated with improvement in endothelial function. In the analysis plasma levels of glucose, insulin, cholesterol, LDL cholesterol, cigarette smoking, hypertension, new use of any cardio-active or antiplatelet or lipid-lowering drug, were entered as independent variables.

Results Clinical features of study patients are shown in Table 1, patients assigned to metformin and placebo had similar male/female distribution and clinical features. Medication compliance was 97% in the metformin group where one patient withdrew from the study for gastrointestinal side-effects and 91% in the placebo group where three patients withdrew because of the complaint of weight gain (one patient), nausea (one patient) and unwillingness to continue the study (one patient). At baseline all subjects had similar values of endothelial function (FMD 7.4 ± 2.1% vs. 7.3 ± 2.5%) and value of HOMA-IR (3.39 ± 1.2 vs. 3.42 ± 1.5). After 3 months no significant differences in blood pressure or BMI were detected between groups. A significant improvement in insulin resistance was noted in patients receiving metformin compared with those allocated to placebo (HOMA-IR from 3.39 to 2.5 vs. form 3.42 to 3.37;


2005 Blackwell Publishing Ltd Journal of Internal Medicine 258: 250–256

METFORMIN IN METABOLIC SYNDROME

Table 1 Clinical characteristics of study patients Patients Metformin Placebo (65) (32) (33) Mean age (years) Men/women BMI Risk factors for CAD (%) Cigarette smoking Dyslipidaemia Hypertension Grade 1–2 IR/IGT/diabetes Family history for CAD Aspirin/ticlopidine/clopidogrel Anticoagulants Diuretics b-Blockers Ca-antagonists ACE-I ARB Statins

54 ± 6 37/28 32 ± 3

55 ± 4 18/14 31 ± 5

54 ± 7 19/14 32 ± 4

14 100 100 76 100 36 47 3 43 36 19 28 33 43

16 100 100 72 100 31 22 2 23 20 9 14 15 20

12 100 100 79 100 39 25 1 20 16 10 14 18 23

Controls were more frequently smokers and had family history of CAD but had less frequently hyperlipidaemia or arterial hypertension. Use of drugs at randomization in patients allocated to metformin and placebo. No significant difference in the use of any class of drug was found. BMI, body mass index; IR, insulin resistance; IGT, impaired glucose tolerance; CAD, coronary artery disease; ACE, angiotensin converting enzyme; ARB, angiotensin receptor blockers.

26% reduction in HOMA-IR, P < 0.001). Also a decrease in plasma fasting glucose levels was detected in patients taking metformin but not in those on placebo (from 123 ± 5 to 108 ± 3 mg dL)1 vs. 121 ± 6 to 122 ± 7 mg dL)1; Table 2). Compared with baseline and placebo patients who received metformin had significant improvement of endothelial function (FMD 12.4 ± 1.9% vs. 6.9 ± 2.7%; metformin vs. placebo, P ¼ 0.0016; Fig. 1), whereas no significant effect Table 2 Baseline clinical characteristics of study patients and controls

was seen on endothelium-independent response to sublingual glyceryl trinitrate (P ¼ 0.32). A significant positive correlation was found between the improvement in insulin resistance and that in endothelial function (r ¼ )0.58, P ¼ 0.0016; Fig. 2). No correlation was found between the improvement in fasting plasma glucose and endothelial function. The multivariate analysis showed the improvement in insulin resistance as the only parameter significantly associated with the improvement in endothelial function.

Discussion The present study shows that 3 months of treatment with metformin improves both insulin resistance, measured by HOMA-IR, and endothelial function in patients with MS. Our data are in agreement with a previous study of Mather et al. in which metformin improved endothelium-dependent vascular response and insulin sensitivity in patients with type 2 diabetes without other metabolic abnormalities. More recently De Jager et al. [20] have shown that in patients with type 2 diabetes treated with insulin, metformin treatment was associated with improvement of endothelial function, which was not related to changes in glycaemic control. In addition our results show that the improvement of endothelial function observed with metformin is directly linked to the improvement of insulin resistance and not related to the improvement in fasting plasma glucose suggesting the prominent role of insulin resistance in the pathogenesis of endothelial dysfunction in patients with MS [21]. Metabolic syndrome is a clustering of metabolic risk factors whose importance is increasing as one of the major issue in the management of cardiovascular Metformin

HOMA-IR Glucose (mg dL)1) HbA1c (%) Cholesterol (mg dL)1) LDL-c (mg dL)1) HDL-c (mg dL)1) Triglycerides (mg dL)1)

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Placebo

Baseline

Follow-up

Baseline

Follow-up

P value

3.39 123 ± 5 5.4 ± 0.7 231 ± 27 168 ± 11 31 ± 7 243 ± 21

2.5 108 ± 3 5.1 ± 0.9 218 ± 31 146 ± 14 37 ± 9 219 ± 18

3.42 121 ± 6 5.6 ± 0.8 247 ± 33 174 ± 16 30 ± 8 236 ± 15

3.37 122 ± 7 5.6 ± 1.3 240 ± 29 165 ± 12 32 ± 12 247 ± 26