Chronic systemic inflammation is associated with the development of several prevalent age- and inactivity- related diseases, such as atherosclerosis and diabetes (1,15,32). There are many biomarkers of systemic inflammation, with interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1β), and C-reactive protein (CRP) the most frequently measured (31,33).
Several researchers have shown that inflammatory cytokines play a major role in inducing CRP production. Hepatocyte CRP expression and production for example, is enhanced by IL-6 and TNF-α exposure (25,40). Moreover, inflammatory cytokine-induced CRP production has been observed in human coronary artery smooth muscle cells suggesting that local production of CRP in the vessel may directly influence the development of atherosclerosis (6).
Elevated blood concentrations of inflammatory cytokines and CRP are linked to increasing age (5,30). CRP and IL-6 have been extensively reviewed and are positively associated with increased age in vivo (9,22,30). Numerous therapeutic and pharmacological modalities have been explored in an effort to reduce chronic levels of inflammation associated with aging. Regular exercise appears to significantly lower circulating CRP and inflammatory cytokine concentrations (10,22,36). Ruben et al. found that physically active individuals had lower plasma concentrations of IL-6 and TNF-α when compared to age- and gender- matched inactive groups (28). Several researchers have also reported a negative correlation between physical activity level and CRP (2,27). These studies are cross-sectional in nature and an intervention may provide more insight into the effectiveness of exercise as an anti-inflammatory agent.
Recent evidence has provided substantial support for exercise intervention-induced reductions in circulating markers of inflammation (10,22). Okita et al. found that a two-month aerobic exercise program significantly reduced CRP from 0.63 to 0.41 mg·L−1 in 199 older women (22). However, a number of studies have shown that exercise programs do not influence these markers of inflammation (11,17,20). A recently published meta-analysis concluded that aerobic exercise training did not significantly alter circulating levels of CRP (12). The randomized, controlled trials highlighted in this work utilized an aerobic exercise intervention and the intensity of the exercise was variable, ranging from 40-80% of maximal oxygen uptake (12). Despite these findings, emerging data supports the ability of resistance training to reduce circulating levels of CRP in diseased populations (4,7,38); however, there is no consensus on the influence of a program involving resistance training on CRP levels in healthy individuals. Consequently, there is a need for concrete evidence either supporting or refuting the use of resistance exercise as an intervention in reducing inflammation in an able-bodied population. The purpose of this study was to examine the effect of age, physical activity level and a 12-wk aerobic and resistance exercise training program on serum CRP and plasma IL-6, TNF-α and IL-1β.
Twenty-nine younger (18-35 yr) and 31 older (65-85 yr) subjects were assigned to one of four groups according to age and physical activity level: young physically active (YPA, N = 15; 25 ± 5 yr), young physically inactive (YPI, N = 14; 25 ± 4.7 yr), old physically active (OPA, N = 14; 71 ± 4 yr), or old physically inactive (OPI, N = 17; 71 ± 4 yr) groups (Table 1). Even though this study was not designed to examine a gender effect, relatively equal numbers of men and women were recruited into each group to satisfy Purdue University institutional review board requirements (Table 1). The Paffenbarger questionnaire (23) and the modified Balke submaximal V˙O2max test (3) were used to assign subjects to the appropriate physical activity group (physically active or physically inactive) (34). Briefly, participants with a "high" to "very high" level of activity and "good" to "excellent" estimated V˙O2max (YPA group females, > 38 mL·kg−1·min−1; YPA group males, > 45 mL·kg−1·min−1; OPA group females, > 28 mL·kg−1·min−1; OPA group males, > 35 mL·kg−1·min−1) were enrolled into the physically active (PA) groups. Subjects with physical activity scores in the "low" to "very low" category and estimated V˙O2max in the "below average" category (YPI group females, < 31 mL·kg−1·min−1; YPI group males, < 37 mL·kg−1·min−1; OPI group females, < 23 mL·kg−1·min−1; OPI group males, < 26 mL·kg−1·min−1) were placed into the physically inactive (PI) groups. All study participants were observed before and after a 12-wk period. The physically inactive subjects (YPI and OPI) completed a 12-wk combined aerobic and resistance training protocol. The physically active subjects (YPA and OPA) served as controls and continued their regular physical activity programs.
Only healthy subjects were recruited into the study (34). The menstrual regularity of the young women was determined during the screening process, and participating young women were tested on days 5-7 of their menstrual cycle. This study was approved by the committee on the use of human research subjects at Purdue University (ref #02-587).
Participants were asked to obtain permission from their personal physician to participate in the study and sign an informed consent document. In addition, subjects enrolled in the training phase of the study underwent a physical examination conducted by the study physician, completed a treadmill stress test and an eight-repetition maximum (8RM) leg press exercise to ensure that the participant could safely handle the cardiovascular and muscular stressors involved in the study.
Before and after the intervention period, all study participants underwent a series of anthropometric measurements including, height, weight, and body composition, which was determined by bioelectrical impedance (Omron Healthcare Inc, Vernon Hills, IL).
Acclimation and exercise training.
After both personal physician and study (OPI and YPI groups) clearance, participants were acclimated to the treadmill (Lifefitness Treadmills, Schiller Park, IL) and then eight resistance exercises: leg extension, leg flexion, leg press, hip adduction, hip abduction, chest press, seated row, and "lat" pull down (Keiser, Fresno, CA) during a three-session acclimation week.
Each session of the 12-wk training program (3 d·wk−1) involved a warm-up, aerobic training (20 min), resistance training (two sets of eight exercises), stretching, and cool-down period. Aerobic training consisted of treadmill walking/jogging for 20 min at 70-80% of heart rate reserve. The resistance exercise portion (70-80% 1RM) of each workout consisted of an initial set of eight repetitions followed by a second set performed to "momentary muscular failure." The participants were reevaluated on a biweekly basis, and the intensity of the exercise was adjusted accordingly (34). The active control groups (YPA and OPA) were asked to maintain their current level of activity for the 12-wk intervention period.
The postintervention period assessment was performed 4 d after the completion of the training period and included the measurement of estimated V˙O2max and body composition, 8RM and 1RM for resistance exercises, and physical activity levels in all PA and PI subjects. YPA and OPA subjects refrained from exercise during the 4 d before the poststudy testing. The PA groups did not complete strength assessments.
Resting blood samples, obtained at baseline and after the intervention from all subjects, were collected after a 24-h dietary control and 8-10 h of fasting (34). Subjects reported to the lab between 0600 and 0800 h and were seated for 30 min before venous blood samples (40 mL total) were drawn into evacuated tubes containing EDTA, sodium heparin, or SST clot activator (Becton-Dickinson, Franklin Lakes, NJ).
Undiluted plasma was used to determine the concentrations of IL-6, TNF-α and IL-1β using an Enzyme Linked Immunosorbant Assay (ELISA) (e-Bioscience, San Diego, CA). Each kit employed a solid-phase sandwich ELISA specific for IL-6, TNF-α, and IL-1β. Plates were read on a microplate reader using absorbance (Bioteck instruments Inc., Winooski, VT). Intra- and interassay coefficients of variation were below 11%.
C-reactive protein analysis.
According to manufacturer's instructions, serum samples were diluted 1:100 in assay diluent before performing the assay. C-reactive protein was quantified using a commercially available, high-sensitivity, solid-phase sandwich ELISA (ALPCO Diagnostics). Plates were read using a microplate reader (Bioteck instruments Inc., Winooski, VT). Intra- and interassay coefficients of variation were less than 3%.
Descriptive statistics including the mean, median, and standard deviation were calculated for age, height, body mass, percent body fat, BMI, estimated V˙O2max, 8RM and 1RM measurements, plasma IL-1β, IL-6, and TNF-α, and serum CRP. Data were evaluated using a 2 × 2 × 2 (age × physical activity × time) factor ANOVA. The first factor had two levels of age (young or old), the second factor had two levels for physical activity (active or inactive), and the third factor was the repeated measure of time (pre or post). The residuals were checked for normality (Shapiro-Wilkes) using SAS (Cary, NC). To stabilize the variance, log transformations were applied to weight, estimated V˙O2max, serum CRP, and body fat percentage. Significance was set at P < 0.05 and t-tests with a Bonferroni multiple comparison correction were used post hoc. Pearson stepwise correlation analysis was used to determine relationships between all measurements. All values are presented as the mean ± standard error (SE).
Subject descriptive data.
As reported previously, there were significant differences among groups for body mass, body mass index, and body fat percentages (Table 1) (34). Baseline cross-sectional analysis revealed that both inactive groups (YPI and OPI) had higher body mass (P < 0.01), BMI (P < 0.001) and body fat percentage (P < 0.0001) when compared with the active controls (YPA and OPA) (Table 1) (34). There was a decrease in BMI and body mass over time (P < 0.001; P < 0.01) in all groups, but there were no significant differences in any of the other anthropometric measures from pre- to postintervention (Table 1) (34).
Training program results.
The older subjects (OPA and OPI) and the physically inactive subjects (YPI and OPI) had significantly lower estimated V˙O2max at baseline compared with younger subjects (YPI and YPA) and physically active subjects (YPA and OPA), respectively (Fig. 1) (34). Furthermore, a physical activity × time interaction revealed that those individuals enrolled in the training program had a significant, 10.4% increase in their estimated V˙O2max compared with controls (P < 0.01) (Fig. 1) (34). Estimated V˙O2max still remained higher in the PA groups compared with the PI groups (P < 0.01) after the intervention (Fig. 1). There were also significant strength differences between YPI and OPI at the start of the study (P < 0.001) for all exercises; however, both OPI and YPI had significant strength gains ranging from 14.4 to 58.7%, with a 38.1% average increase, in all exercises during the course of the 12-wk training period (P < 0.01) (34).
Serum CRP concentrations.
There was a trend toward higher CRP in inactive subjects; however, there were no significant differences in serum CRP among the groups with respect to age or physical activity level at baseline (Fig. 1). The YPI and OPI groups had a significant decline in serum CRP from pre- to posttraining (P < 0.01) (Fig. 2). Collectively, the change in serum CRP, from pre- to posttraining, was 58% lower for the OPI and YPI groups combined. Furthermore, 25% of the YPI subjects and 52% of the OPI participants had CRP levels > 1 mg·L−1 before training, respectively. These proportions were reduced after the intervention to 14% in the YPI and 11% in the OPI groups. In addition, serum CRP levels after training were not different from those of the YPA and OPA groups (Fig. 2).
Plasma TNF-α, IL-6, and IL-1β.
There were no age or physical activity differences in plasma IL-6 or IL-1β concentrations among the groups at baseline; however, YPA and YPI had higher plasma TNF-α concentrations compared with the older subjects at baseline and after the intervention period (OPA and OPI; P < 0.01; Figs. 3-5). There were no significant intervention effects on plasma IL-6 or IL-1β concentrations among groups (Figs. 4 and 5).
Significant strength and cardiorespiratory fitness improvements and a significant decrease in serum CRP concentration were observed after the 12-wk exercise training intervention. The 58% reduction in serum CRP is particularly intriguing, because most studies have found no change in serum CRP as a result of aerobic exercise training (12). Individuals with CRP concentrations less than 1 mg·L−1 are considered to be at low risk for cardiovascular disease (CVD), whereas individuals with values from 1-3 mg·L−1 and greater than 3 mg·L−1 are at moderate and high risk for CVD, respectively (24). Before training, average serum CRP concentrations in YPI (1.20 mg·L−1) and OPI (1.16 mg·L−1) were in the moderate-risk category, and after the training intervention, average values (YPI: 0.51 mg·L−1, OPI: 0.49 mg·L−1) were in the low-risk category. A training-induced reduction in CRP is supported by a few similar investigations (18,22). On the other hand, many interventions have shown less of a decrease, which may be attributable to the fact that this study used a supervised, progressive aerobic and resistance exercise intervention period.
In other investigations, when active individuals were compared with inactive individuals, higher levels of physical activity have been associated with a lower concentration of serum CRP (2,27,28). In the present study, there were no significant relationships between CRP and physical activity level (data not shown); however, in a larger cross-sectional comparison of similar groups (active, inactive, old, and young), both the older and inactive groups, regardless of age, had significantly higher CRP (19).
There are established relationships among CRP, BMI, fat mass, and percent body fat (13). Although our results support the positive relationship between body mass and CRP, it has been hypothesized that body mass reduction may result in decrements in CRP concentration. Even though there were significant improvements in estimated V˙O2max and muscular strength after training, there were no training-related changes in body mass, BMI, or body fat percentage (34). Because the primary aim of this study was to use exercise to reduce inflammation, the intervention period was 12 wk in duration. Longer intervention periods (> 20 wk) have been found to be more effective in reducing BMI and body fat percentage (39). Because there is evidence linking changes in CRP to alterations in body fat percentage (8,35), it was somewhat surprising to observe a significant reduction in serum CRP concentration without a reduction in body fat percentage in the training groups. These findings are supported by a study that examined the CRP concentrations in 67 ultramarathon runners and 63 sedentary, male controls and found that after adjustment for BMI, there was only a modest difference in CRP between the runners and controls (37). It is important to note that bioelectrical impedance is not the best measure of body composition. The measurement of body composition in this study was intended to be descriptive in nature and has been used in other studies with some accuracy (29). These results, coupled with our study findings, highlight the possibility that factors other than a decrease in body mass/body fat percentage may be linked to lower CRP concentrations.
The mechanism of an exercise training-induced reduction in CRP remains unknown. Some have hypothesized that the changes in circulating inflammatory cytokine concentrations may lead to alterations in hepatic CRP production (26); however, our study failed to reveal an effect of an exercise training intervention on resting, fasting plasma IL-6, TNF-α, or IL-1β concentration. Interestingly, there is support for either exercise-induced reductions (7) or unchanged levels (38) of plasma/serum inflammatory cytokines. Because most of these studies involve diseased populations, it is possible that the response to exercise training may be more robust and easier to detect than in healthy individuals. Additionally, even though significant alterations in resting inflammatory cytokines were not detected in this study, it is possible that the repeated, significant and transient changes in IL-6 levels observed in acute, intense exercise bouts (26) may act to downregulate CRP protein production in the liver. These alterations could possibly result in lower levels of circulating CRP in trained compared with untrained individuals.
Older individuals (OPA and OPI) in the present study had lower baseline plasma TNF-α concentration than younger subjects (14). Although the reason for these deviations from the established literature is currently unknown, a further exploration of the link between inflammation and exercise/diet may help provide an explanation (16,21).
In conclusion, a combined aerobic and resistance exercise training program may serve as a promising therapeutic modality resulting in a decline of serum concentration of CRP. Future studies should focus on the mode and the mechanism by which exercise reduces CRP.
This study was funded by the American Heart Association (AHA Grant # 0350612Z). In addition, we would like to thank the subjects for participating in this project. Furthermore, we acknowledge the technical assistance of Nadine Carnell and Janet Green in the control diet development and Dr. Michael Krauss for medical support.
There were no conflicts of interest associated with this study.
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