Weight Loss Reduces Plasma Endothelin-1 Concentration in Obese Men

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Seiji Maeda*1,
Subrina Jesmin*,
Motoyuki Iemitsu*,
Takeshi Otsuki*,
Tomoaki Matsuo,
Kazunori Ohkawara,
Yoshio Nakata*,
Kiyoji Tanaka*,
Katsutoshi Goto and
Takashi Miyauchi*

^* Center for Tsukuba Advanced Research Alliance, Institute of Health and Sport Sciences, Cardiovascular Division, Institute of Clinical Medicine, and Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan

¹ Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail: smaeda{at}tara.tsukuba.ac.jp

Obesity is associated with endothelial dysfunction that may contribute to the development of diabetes, hypertension, and atherosclerosis. Endothelin-1 (ET-1), which is produced mostly by vascular endothelial cells, has potent vasoconstrictor and proliferative activity in vascular smooth muscle cells and, therefore, has been implicated in regulation of vascular tonus and the progression of atherosclerosis, suggesting that ET-1 may be important in endothelial dysfunction. We studied whether diet-induced weight loss (i.e., lifestyle modification) affects plasma ET-1 concentration in obese individuals. We measured plasma ET-1 concentration in seven obese men (age: 48 ± 4 years old, body mass index: 27.7 ± 0.5 kg/m²) before and after a 3-month, diet-induced weight reduction program (i.e., lifestyle modification program). Caloric restriction reduced body weight from 78 ± 3 to 68 ± 2 kg (P < 0.001) and resulted in 12.1 ± 1.2% reduction in body mass index (24.3 ± 0.3 kg/m², P < 0.0001). After the weight reduction program, systolic and diastolic blood pressure significantly decreased (128 ± 7 vs. 115 ± 4 mm Hg, P < 0.05 and 88 ± 4 vs. 77 ± 2 mm Hg, P < 0.01, respectively). The plasma level of ET-1 significantly decreased after the program (5.1 ± 0.4 vs. 4.0 ± 0.3 pg/ml, P < 0.05). The percentage systolic blood pressure reduction and percentage plasma ET-1 concentration reduction was in a linear relationship (r = 0.86, P < 0.05). Furthermore, the relationship between percentage weight reduction and percentage plasma ET-1 concentration reduction was linear (r = 0.87, P < 0.05). We conclude thatweight loss by low-calorie diet (i.e., lifestyle modification)reduces plasma ET-1 concentration in obese individuals. Thisreduction may contribute to the improvement of obesity-inducedendothelial dysfunction.

Keywords: endothelin-1, obesity, endothelial dysfunction, diet

Obesity is strongly associated with endothelial dysfunction that may play a role in the development of diabetes, hypertension, and atherosclerosis (1, 2). Endothelial dysfunction is a common abnormality in obesity. Damage to the endothelium is an important risk factor for cardiovascular diseases because it leads to structural changes, such as thickening of the intima and media of vessel walls. Clinical and animal studies have confirmed a strong relationship between obesity and cardiovascular disease such as diabetes, hypertension, and atherosclerosis (2–4). However, mechanisms linking obesity with endothelial dysfunctionhave not yet been fully clarified.

Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis in health and diseases (5, 6). Endothelin-1 (ET-1) is a potent vasoconstrictor peptide produced by vascular endothelial cells (6, 7). ET-1 has potent vasoconstrictor effect on vascular smooth muscle cells (8). It has also been reported that systemic administration of an endothelin receptor antagonist significantly decreased systemic blood pressure and peripheral vascular resistance in healthy humans, strongly suggesting that endogenous generated ET-1 contributes to basal vascular tonus in humans (9). Furthermore, ET-1 is a pro-mitogen, potentiating the response of other growth factors; therefore, ET-1 has been implicated in the progression of atherosclerosis (6, 10, 11).Thus, ET-1 has been implicated in regulation of vascular tonusand progression of atherosclerosis, suggesting that ET-1 maybe important in endothelial dysfunction.

Obesity is strongly associated with endothelial dysfunction.However, the mechanisms underlying obesity-induced endothelialdysfunction are unclear. Endogenous ET-1 may play an importantrole in endothelial dysfunction because ET-1 has been implicatedin regulation of vascular tonus and progression of atherosclerosis.The purpose of the present study was to examine whether diet-inducedweight loss affects plasma ET-1 concentration in obese individuals.It is of great interest and importance to study whether weightloss causes a decrease in plasma ET-1 concentration in obesehumans. We hypothesized that weight loss can reduce plasma ET-1concentration in obese individuals, and that this reductioncontributes to the improvement of obesity-induced endothelialdysfunction. In the present study, we measured plasma ET-1 concentrationin obese men before and after a 3-month, diet-induced weightreduction program.

Subjects.

Seven obese men participated in the study (48 ± 4 years old, height: 167.6 ± 2.0 cm, body mass index [BMI]: 27.7 ± 0.5 kg/m²). None of the participants was taking medicationon a regular basis at the time of the study. The obese subjectsperformed a 3-month, low-calorie diet intervention study.

The study was approved by the Ethical Committee of the Instituteof Health and Sport Sciences, the University of Tsukuba. Thisstudy conformed with the principles outlined in the HelsinkiDeclaration, and all subjects gave their written informed consentbefore inclusion in the study.

Experimental Design.

All obese men were studied before and after 3 months of diet-inducedweight reduction program (i.e., lifestyle modification program).Body weight, BMI, systolic blood pressure, diastolic blood pressure,venous plasma glucose concentration, and venous plasma ET-1concentration were measured before and after a 3-month, diet-inducedweight reduction program in the obese men. Before they weretested, subjects fasted for 12 hrs. Blood pressure was measuredin duplicate, with subjects in the upright sitting position.All measurements were performed at a constant room temperature(25°C).

Dietary Protocol.

All subjects were instructed to take meals per day consistingon average of 420 kcal of protein, 840 kcal of carbohydrate,and 420 kcal of fat (total: 1680 kcal/day). Subjects kept dailyfood diaries during the 3-month intervention period and learnedabout proper daily nutrition (well-balanced protein, carbohydrates,fat, various amino acids, vitamins, and minerals) through weeklylectures and counseling by skilled dieticians.

Measurement of Plasma ET-1 Concentration by Sandwich-Enzyme Immunoassay.

Each blood sample was placed in a chilled tube containing aprotinin (300 KIU/ml) and EDTA (2 mg/ml), and was then centrifuged at 2000 g for 15 min at 4°C. The plasma was stored at –80°C until use. Plasma (1 ml) was acidified with 3 ml of 4% acetic acid, and immunoreactive ET-1 was extracted with a Sep-Pak C18 cartridge (Waters, Milford, MA), as previously described in our article (12). The elutes were reconstituted with 0.25 ml of assay buffer and were subjected to sandwich-enzyme immunoassay. The sandwich-enzyme immunoassay for ET-1 was carried out as previously described using immobilized mouse monoclonal antibody AwETN40, which recognizes the NH₂-terminal portion of ET-1, and peroxidase-labeled rabbit anti-ET-1 COOH-terminal peptide (15–25) Fab’ (12). The Fab’ fragment of this rabbit antibody was used as an enzyme-labeled detector antibody after being coupled with horseradish peroxidase. The coefficient of variation of the ET-1 assay for intraassay variation was 11% and coefficient of variation for interassay variation was 13% (13). We previously reported that the lowest detection limit of this assay was 0.4 pg/ml for ET-1 (14). The plasma ET-1 levels in the present studywere far beyond the lowest limit of detection with this assay(0.4 pg/ml) in all subjects.

Statistics.

Values are means ± SE. To evaluate differences in the levels before and after weight reduction program in obese men, Student’s t test for paired values was used. P < 0.05was accepted as significant.

All seven obese men completed the 3-month, low-calorie diet intervention study. Table 1 shows the body weight, BMI, blood pressure, and plasma glucose concentration in the obese men before and after 3 months of the weight reduction program. Body weight markedly decreased after the weight reduction program (Table 1). BMI also remarkably decreased after the program (Table 1). After the weight reduction program, systolic blood pressure and diastolic blood pressure significantly decreased (Table 1). There was no significant change in plasma glucose concentration before and after the program (Table 1). The plasma level of ET-1 significantly decreased after the program (5.1 ± 0.4 vs. 4.0 ± 0.3 pg/ml, P < 0.05; Fig. 1). Figure 2 shows the relationship between the percentage plasma ET-1 concentration reduction and the percentage systolic blood pressure reduction or percentage weight reduction. The percentage systolic blood pressure reduction and percentage plasma ET-1 concentration reduction was in a linear relationship (r = 0.86, P < 0.05; Fig. 2A). Furthermore, the relationship between percentage weight reduction and percentage plasma ET-1 concentration reduction was linear (r = 0.87, P < 0.05; Fig. 2B).

In the present study, we measured plasma ET-1 concentrationin obese men before and after a 3-month, diet-induced weightreduction program. After the program, because of which the bodyweight and BMI markedly decreased, the plasma ET-1 concentrationsignificantly decreased. We also demonstrated that the low-caloriediet in obese men significantly decreased systolic and diastolicblood pressure, suggesting the improvement of endothelial dysfunction.Furthermore, there was a significant positive correlation betweenthe percentage plasma ET-1 concentration reduction and percentagesystolic blood pressure or body weight reduction in obese men.Therefore, we suggest that the reduction of ET-1 by weight lossmay contribute to the improvement of obesity-induced endothelialdysfunction because ET-1 has been implicated in regulation ofvascular tonus and progression of atherosclerosis.

Obesity is strongly associated with cardiovascular disease (1–4). Obese individuals are at increased risk for diabetes, hypertension, atherosclerosis, and other cardiovascular diseases. Obesity is also associated with endothelial dysfunction that may play a role in the development of hypertension and atherosclerosis (1). Endothelial dysfunction is a common abnormality in obesity. Vascular endothelial cells produce ET-1, which is a potent vasoconstrictor peptide and has potent proliferating activity in vascular smooth muscle cells (6, 7, 9–11). Thus ET-1 has been implicated in regulation of vascular tonus and progression of atherosclerosis, suggesting that ET-1 may be important in the endothelial dysfunction. Cardillo and colleagues (15) have shown recently that blockadeof endothelin A receptor induces significant vasodilation inoverweight and obese humans. Therefore, increased vascular productionof ET-1 in hypertensive patients with increased body mass hasbeen suggested as a potential mechanism for endothelial dysfunction.In the present study, the plasma ET-1 concentration significantlydecreased after the weight reduction program. The present studyalso demonstrated that blood pressure reduced after the program.We considered that the reduction of ET-1 may participate inthe mechanism underlying the improvement of endothelial dysfunctionby modest weight loss. Therefore, it is possible that modestweight loss can improve endothelial dysfunction in obese humansthrough reduction of ET-1 production.

Several reports indicate that weight loss and lifestyle modifications can improve endothelial function. It has been shown that 6 months of weight reduction and exercise improve endothelial function and reduce selective markers of endothelial activation and coagulation in obese individuals (16). Thus lifestyle modification in the form of caloric restriction and moderate intensity physical exercise in obese subjects may be of importance for improvement of endothelial dysfunction. A recent study demonstrated that weight reduction with a very low-calorie diet improves flow-mediated vasodilation in obese subjects (17). It has been reported that, after 2 weeks of low-calorie intake, a significant improvement in flow-mediated dilatation was observed in obese hypertensive patients (18). Furthermore, healthy premenopausal obese women after 1 year of a weight reduction progam were able to reduce body weight (10%) with an improvement in vascular responses to L-arginine (19, 20). The present study demonstrated thatthe plasma ET-1 concentration significantly decreased afterthe weight loss program with reduction of blood pressure. Therefore,we suggest that weight loss (i.e., lifestyle modification) reducesplasma ET-1 concentration in obese men, and this reduction mayparticipate in improvement of endothelial dysfunction, therebycontributing to beneficial effects on the cardiovascular system(i.e., prevention of progression of hypertension and atherosclerosisby endogenous ET-1).

The plasma ET-1 concentration in healthy humans was 1.0–1.5 pg/ml (12), and significantly increased with aging (i.e., plasma ET-1 concentration was markedly higher in healthy older humans than in healthy young or middle-aged humans) (12). Our laboratory previously reported that plasma ET-1 concentration is increased in some human diseases (14, 21) (e.g., chronic heart failure[22] and acute myocardial infarction [21]). In the present study,the plasma ET-1 concentration in obese humans was 5.1 ±0.4 pg/ml, and was clearly higher in obese humans than in healthyhumans. Therefore, the increased plasma ET-1 concentration maybe associated with obesity-induced diabetes, hypertension, atherosclerosis,and other cardiovascular diseases.

In conclusion, we demonstrated that weight reduction in obesemen significantly decreased plasma ET-1 concentration. BecauseET-1 has potent vasoconstrictor and proliferative activity invascular smooth muscle cells and has been implicated in theregulation of vascular tonus and the progression of atherosclerosis,we propose that the decrease in production of ET-1 by weightloss may be partly involved in the improvement of endothelialdysfunction in obese individuals.

Supported by grants-in-aid for scientific research from theMinistry of Education, Science, Sports and Culture of Japan(16500391), and a grant from the project of Tsukuba AdvancedResearch Alliance (TARA) at the University of Tsukuba.

Received for publication September 28, 2005.

Accepted for publication November 10, 2005.

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