a Department of Medicine, Nordland Hospital, Bodø, Norway
b Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway
c Rønvik Health Center, Bodø, Norway
d Research Institute of Internal Medicine, Department of Cardiology, Rikshospitalet University Hospital, Oslo, Norway
e Department of Clinical Chemistry, Rikshospitalet University Hospital, Oslo, Norway
f Department of Radiology, Nordland Hospital, Bodø, Norway
g Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway
* Correspondence to: Dr K. T. Lappegard, Department of Medicine, Nordland Hospital, Prinsens gt. 164, N-8092 Bodø, Norway. Tel: +47-755 34000; Fax: +47-75534742
E-mail address: knut.lappegard{at}nlsh.no
Received 16 April 2003; revised 13 November 2003; accepted 4 December 2003
Abstract
Aims Inflammation plays an essential role in the atherosclerotic process, and chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) seem to play a pivotal role in the pathogenesis of atherosclerosis. A possible common inflammatory basis for the pathogenesis of type 2 diabetes, metabolic syndrome and atherosclerosis has been suggested. In this study we investigated the effect of physical exercise and the HMG-CoA reductase inhibitor pravastatin on peripheral markers of inflammation in subjects with the metabolic syndrome.
Methods The study was an unmasked randomized 2x2 factorial trial of 12 weeks duration.
Results In the combined exercise groups there was a significant reduction in MCP-1 and IL-8 of 48pg/ml (P=0.04) and 1.0pg/ml (P=0.007), respectively, as compared to the combined non-exercise groups. There was also a significant reduction vs baseline of 50pg/ml (33%) (P=0.002) and 0.35pg/ml (13%) (P=0.03) for MCP-1 and IL-8, respectively. Changes in MCP-1 were significantly correlated to changes in visceral fat (r=0.41, P=0.02).
Conclusion The protective effect of exercise might in part be due to suppression of the inflammatory process.
Key Words: Atherosclerosis Chemokines Exercise Inflammation Metabolic syndrome Visceral fat
1. Introduction
The metabolic syndrome, or the insulin resistance syndrome, is a common precursor to type 2 diabetes. It is characterized by the simultaneous presence of several cardiovascular risk factors, including abdominal obesity, dyslipidaemia (elevated triglycerides, low HDL cholesterol), hypertension and insulin resistance in the presence or absence of overt glucose intolerance.1Type 2 diabetes is recognised as a major cardiovascular risk factor,2and is associated with increased frequency of conventional risk factors for cardiovascular disease.3The metabolic syndrome, which is much more prevalent than type 2 diabetes, is also considered a major risk factor in recent guidelines.1
It has gradually become evident that inflammation plays an essential role in atherosclerosis. Leukocyte infiltration into the vascular wall is involved in virtually all stages of the process, from the fatty streak to the complex atheromatous plaque.4Increasing evidence suggests that chemokines, such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), play an important role in this process, in part by attracting mononuclear cells to the arterial wall.5Moreover, C-reactive protein (CRP) has been identified as a negative prognostic factor for the development of coronary heart disease both in healthy individuals6,7and in subjects with the metabolic syndrome,8and plasma levels of CRP may be modified by HMG-CoA reductase inhibitors (statins), independent of the LDL-lowering effect of these drugs.9Furthermore, complement activation seems to be involved in both initiation and progression of atherosclerosis.10Indeed, some investigators have pointed out that both type 2 diabetes, metabolic syndrome and atherosclerosis are multifactorial conditions which appear to have a common inflammatory basis.11
Adipose tissue is metabolically active and secretes various chemokines12and proinflammatory cytokines such as interleukin-6 (IL-6)13and tumour necrosis factor- (TNF-
).14Endurance training15and strength training16have both been shown to decrease the amount of visceral fat, and endurance training has beneficial effect on several components of the metabolic syndrome.17Given that a combination of endurance training and strength training will decrease the amount of visceral fat, we sought to determine the effects of such exercise on plasma levels of several inflammatory mediators and on the components of the metabolic syndrome. Pravastatin was used for comparison, because of its documented anti-inflammatory effects.9,18
2. Methods
2.1. Experimental design and participants
The study was an unmasked randomized 2x2 factorial trial of 3 months duration. The participants were recruited after an advertisement in the local newspaper. The inclusion criteria were: men aged 2075 years with the metabolic syndrome (as defined in ref.1), physically inactive (<30min of physical activity per day) and with central obesity (as central obesity is not mandatory for the definition of the metabolic syndrome but of particular interest in this study, a waist circumference >90cm, which is associated with increased cardiovascular risk,19was defined as the cut-off). Individuals with known heart disease were excluded. Those treated with statins were either excluded, or, for four subjects where it was considered medically safe, statins were washed out during a period of 4 weeks and the persons included.
During a period of 2 months, 34 participants were selected out of a total of 110 screened. Baseline characteristics are shown in Table 1. It should be noted that 21 of the participants had diabetes. The participants were randomized into one of four groups: control (group C, n=6), exercise only (group E, n=9), pravastatin only (group P, n=9) or the combination of pravastatin and exercise (group PE, n=10). Written informed consent was obtained.
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The study was approved by the regional ethics committee and conformed with the principles outlined in the Declaration of Helsinki.
2.2. The intervention programme
The exercise programme consisted of endurance type of exercise, such as walking/jogging on treadmill, and strength training. Workouts were performed three times a week in a training studio, mostly in supervised groups. The duration of each workout was 4560min. Approximately 40% of the scheduled workout was walking/jogging/cycling and 60% was strength training. The strength training was performed in cycles with 1520 repetitions per cycle, and large muscle groups such as thighs, back and abdomen were trained. The participants were encouraged to record the training in logs.
Participants in the pravastatin group received 40mg pravastatin per day, a dosage known to exert anti-inflammatory effects in vivo,9as well as clinical benefit in both primary20and secondary21,22prevention of coronary heart disease. Groups E and PE were not separated during physical exercise. Participants in groups C and P were encouraged not to change their life style during the intervention period.
2.3. Laboratory procedures
Blood pressure was measured in the sitting position after 5min rest. Three recordings were made, and the mean of the last two measurements was used for statistical analysis.
Oxygen uptake was measured during walking/jogging on a treadmill at the same heart rate at baseline and after intervention to obtain appropriate comparability. This target heart rate was defined as 80% of maximum, estimated from the equation 220age (years). Five recordings were made at the target frequency, and the median was used for statistical analysis. The expired air was analysed using a SensorMedics system. Gas analysers were calibrated before each test.
Computerized tomography (CT) scans were performed on a General Electric Pace Plus scanner using validated procedures.23The scans were performed at 120kV. The subjects were examined in supine position with their arms stretched above their head. To minimize the amount of radiation, the CT scans were performed at only one site: at the level corresponding to the disk between the 2nd and the 3rd lumbar vertebrae. In men, this single scan has showed a higher correlation with visceral adipose tissue volume determined by multiple scans than any other single scan.23In order to obtain high accuracy and reproducibility for the measurements, a scout view was used to establish the position of the scans to the nearest 2mm. The attenuation interval for adipose tissue calculations was 30 to 190 Houndsfield units. Subcutaneous and visceral fat were calculated by delineating their respective areas with a graph pen. All CT scans were examined at the end of the study and the radiologist was blinded to the randomization.
Blood samples were collected between 8 and 10a.m. after an overnight fast. To minimize the influence of any incidental acute phase reaction, participants were requested not to perform any major physical activity the last 5 days before collections of blood samples. Furthermore, in two individuals sampling was delayed for 23 weeks due to minor infection.
Routine blood analyses were performed in the hospital laboratory using standard equipment and assays. Blood, serum and plasma for analysis of HbA1c, C-peptide, high sensitivity CRP, complement activation products, interleukins, MCP-1 and TNF-a were frozen in aliquots at 70°C and analysed in parallel at the end of the study. The complement activation products C3bc and TCC (terminal SC5b-9 complement complex) were quantified in ELISA assays as previously described.24Interleukins 6, 8 and 10 and TNF- were analysed using PeliKine Compact EIA kits (CLB, Amsterdam, The Netherlands). MCP-1 was determined by EIA with the Duoset from R&D Systems (Minneapolis, MN), according to the manufacturer's instructions.
2.4. Statistics
Non-parametric tests were used for statistical analyses. The 2x2 factorial design was used to compare the combined exercise group E+PE with the combined non-exercise group C+P, and the combined pravastatin group P+PE with the combined non-pravastatin group C+E. Changes from baseline were evaluated in the combined exercise group E+PE and in the combined pravastatin group P+PE, respectively. Inter-group comparisons were evaluated using the MannWhitney U test. Wilcoxon's test for paired observations was used to evaluate changes from baseline within the combined intervention groups. Spearman correlation was used for regression analyses. The chosen significance level was a two-tailed P<0.05.
3. Results
3.1. Anthropometry and physiology
The results are presented in Table 2. In the combined exercise group E+PE, there was a significant increase in exercising oxygen consumption (at 80% of estimated maximum heart rate) with a median of 4.6ml/kg per minute (P=0.007) vs the combined non-exercise group C+P, and of 3.1ml/kg per minute vs baseline (P=0.0006). In the combined exercise group, waist circumference decreased with a median of 8.5cm (P=0.0001) vs the combined non-exercise group, and 7.5cm (P=0.0004) vs baseline. Systolic blood pressure was reduced in the combined exercise group with a median of 6mmHg (P=0.01) vs the combined non-exercise groups and 5mmHg (P=0.0005) vs baseline. Diastolic blood pressure was reduced in the combined exercise group with a median of 4mmHg (P=0.003) vs baseline, and there was a trend towards decrease of 4mmHg (P=0.05) vs the combined non-exercise groups. Body mass index was decreased in the combined exercise group with a median of 1.2kg/m2(P=0.002) as compared to the combined non-exercise group, and 0.7kg/m2(P=0.01) vs baseline.
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3.3. Lipids
The results are presented in Table 3. Pravastatin significantly reduced total cholesterol and LDL-cholesterol, whereas HDL-cholesterol and triglycerides were not affected. In the combined pravastatin group P+PE,cholesterol was reduced with a median of 1.3mmol/l as compared to the combined non-pravastatin group C+E (P=0.0001) and 1.2mmol/l vs baseline (P=0.001). In the combined pravastatin group, LDL-cholesterol was reduced with a median of 1.2mmol/l as compared to the combined non-pravastatin group C+E (P=0.00002) and vs baseline (P=0.001).
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4. Discussion
In this study we investigated the effect of exercise on inflammatory parameters in subjects with the metabolic syndrome. In the combined exercise group E+PE, we observed a statistically significant reduction in both MCP-1 and IL-8 as compared to the combined non-exercise group C+P, and from baseline to end of the observation period.
Strenuous exercise can increase plasma levels of various chemokines and cytokines, including IL-8, IL-6 and TNF- in healthy individuals.25However, epidemiological studies have shown that increasing levels of physical activity are associated with reduced levels of CRP in healthy subjects.26,27Furthermore, in patients with heart failure, who have increased plasma levels of chemokines,28it has been reported that modest exercise can reduce peripheral markers of inflammation, among them MCP-1.29To our best knowledge, our data are the first to show that this also applies to subjects with the metabolic syndrome.
Plasma levels of MCP-1 were also significantly reduced vs. baseline in the combined pravastatin group P+PE. We have recently demonstrated that simvastatin and atorvastatin can reduce gene expression of several chemokines and chemokine receptors in patients with coronary artery disease, although plasma levels of MCP-1 were not reduced.30Inhibition of MCP-1 synthesis by statins in vitro has previously been reported.31To our knowledge, the present study is the first to indicate that statins may decrease plasma levels of MCP-1 in vivo. However, this decrease may in part be a result of the fact that half of the subjects exercised. This notion is supported by the fact that there was no statistically significant difference between the combined pravastatin group P+PE and the combined non-pravastatin group E+C regarding MCP-1. The more extensive lowering of MCP-1 in group PE than in groups E and P (Fig. 1) suggests an additive, suppressive effect of pravastatin and exercise on plasma levels of MCP-1 and further studies with larger groups are warranted to elucidate these relations.
From our data, pravastatin did not seem to affect plasma levels of IL-8. The reduction in the combined exercise group E+EP is therefore most likely caused by exercise alone. A reduction in plasma IL-8 by exercise has to our knowledge previously not been demonstrated.
The association between inflammation and atherosclerosis is well established. In fact, chemokines seem to play a pivotal role in the pathogenesis of atherosclerosis, in part by attracting mononuclear cells to the vessel wall.5Knockout-mice lacking MCP-1 or IL-8 or their corresponding receptors have significantly reduced progression of atherosclerosis.32MCP-1 and IL-8 have been reported to trigger the firm adhesion of monocytes to vascular endothelium under flow conditions.33MCP-1 is also involved in the activation of macrophages in the atheromatous plaque, and plasma levels are elevated in patients with acute myocardial infarction and unstable angina, but not in those with stable angina.34Furthermore, elevated plasma levels of MCP-1 have recently been found to be associated with increased risk of death or myocardial infarction in patients with acute coronary syndromes, and has been identified as a potential therapeutic target.35Thus, in light of our findings, the beneficial effect of exercise in regard to cardiovascularmortality36might at least in part be due to reduction in MCP-1.
Several studies have provided support for the hypothesis that insulin resistance may be associated with chronic subclinical inflammation. High glucose concentrations have been found to induce the release of IL-8 in endothelial and smooth muscle cells, giving one possible explanation for the link between hyperglycaemia, inflammation and atherosclerosis.37CRP, IL-6 and TNF- have been shown to be elevated in association with quantitative measures of insulin resistance.38Adipose tissue is metabolically active and pre-adipocytes have been reported to secrete both MCP-1 and IL-8.12
It seems from our data as if the reduction in chemokines, especially MCP-1, is more correlated to a reduction in visceral fat than to a reduction in subcutaneous fat or increased oxygen consumption (Fig. 2). Although visceral fat did not decrease significantly in the exercise groups, the reduction in especially MCP-1 was significantly correlated to a reduction in visceral fat, irrespective of intervention group, indicating that the reduction in visceral fat, rather than the intervention itself, might influence the plasma levels of chemokines. Whether visceral fat exerts a direct inflammatory effect or whether other mechanisms are involved remains unclear. Both MCP-1 and IL-8 are produced in, and secreted by, endothelial cells, and the production is stimulated by mechanical stretch.39Thus, improved vascular function due to exercise40might in part explain the inhibitory effect on chemokines.
One limitation of our report is the sample size due to the small population in the city where the study was conducted. This will increase the risk of statistically false negative results (type II errors), whereas false positive results (type I errors) are less likely. Thus, we suggest that the data for MCP-1 and IL-8 are the most reliable data obtained in this study. Another limitation of this study is the fact that the control group may have exercised in spite of encouragements not to do so. However, a lack of change in VO2, abdominal circumference, blood pressure and glycaemic control in this group makes this less likely. Moreover, if such unintended exercise really did take place, the statistically significant differences observed between the exercise- and non-exercise groups would be an underestimate rather than an overestimate and in fact underscores the observations. On the other hand, exercise may not have been the only difference between the intervention groups. As the exercise groups, in contrast to the control group and pravastatin group, met on a regular basis, discussions regarding lifestyle changes such as diet may have taken place. The significant reduction in abdominal circumference despite no significant reduction in visceral fat, could have been caused by reduced bowel content due to unintended changes in diet, but may as well be a result of improved bowel function induced by exercise.
Lifestyle changes, including exercise, have been found to reduce the risk of developing diabetes mellitus,41and physical inactivity is an important risk factor for both cardiovascular death and all cause mortality.42Inflammation seems to be involved in the pathogenesis of both diabetes mellitus and atherosclerosis. Our findings suggest an anti-inflammatory effect of exercise through reduced plasma levels of chemokines. The protective effect of exercise might in part be due to suppression of the inflammatory process.
Acknowledgments
The skilful technical assistance of Kari Lise Aakre, Grethe Bergseth, Dorte Christiansen, Gry Edvardsen and Hilde Fure is greatly acknowledged. The contribution from the fitness center S.A.T.S. is highly appreciated. The project was financially supported by the Norwegian Council on Cardiovascular Disease, the Norwegian Foundation for Health and Rehabilitation, the Odd Fellow Foundation and H. Hofstad Berg's Memorial Foundation.
MT, KTL, TC and TEM developed the study protocol. MT was the principal investigator. KTL and TEM were responsible for analysis of cytokines and complement activation products. JKD was responsible for analysis of chemokines. LM contributed with statistical analysis. RB was responsible for quantification of visceral and subcutaneous fat on computerized tomography. All co-authors participated in the writing of the manuscript.
References