Diet and Diet Combined with Chronic Aerobic Exercise Decreases Body Fat Mass and Alters Plasma and Adipose Tissue Inflammatory Markers in Obese Women

The purpose of this study was to investigate the effect of 6 months aerobic exercise and diet alone or in combination on markers of inflammation (MOI) in circulation and in adipose abdominal tissue (AT) in obese women. Thirty obese subjects were randomized into a 24-week intervention: (1) exercise (EX), (2) diet (DI), and (3) exercise and diet (EXD). Blood samples were collected at baseline, after 12 and 24 weeks. AT biopsies were obtained only at baseline and after 24 weeks. In the EXD and DI groups, the fat loss was after 12 weeks was −13.74 and −7.8 % (P < 0.01) and after 24 weeks was −21.82 and −17 % (P < 0.01) with no changes in the EX group. After 12 and 24 weeks, maximal oxygen consumption (VO2max) was increased by 21.81–39.54 % (P < 0.05) in the EXD group and 18.09–40.95 % in the EX group with no changes in the DI group. In the EXD and DI groups, circulating levels of tumor necrosis factor α and interleukin 6 were decreased after 24 weeks for both groups (P < 0.01). No changes in the EX group. Homeostatic model assessment for insulin resistance decreased (P < 0.05) only after 24 weeks in the EXD group. In AT biopsies, subjects in the EXD and DI groups exhibited a significant decrease in MO (P < 0.01 for all). No changes in AT biopsies were found in the EX group. In conclusion, chronic aerobic exercise was found to have no effects on circulating and AT MOI despite an increased VO2max. Rather important body composition modifications were found to have beneficial effects on circulating and AT MOI in these obese women.


INTRODUCTION
The prevalence of obesity has dramatically increased worldwide among adults in recent years [1]. The current World Health Organisation (WHO) estimate is that 1.5 billion adults are overweight worldwide, of which over 500 million individuals are obese. Adipose tissue is found to secrete a variety of inflammatory proteins (adipokines) with autocrine and paracrine effects as well as with systemic effects [2]. Observational studies show that obesity is associated with elevated concentrations of a number of inflammatory proteins such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) [3].
Given these relationships between markers of inflammation (MOI) and adipose tissue stores, a number of investigations have attempted to determine if significant loss of fat mass by diet or by exercise induces a concomitant change in the systemic concentrations of inflammatory mediators.
Several studies have documented that weight loss in conjunction with energy restriction [4,5] or exercise [6,7] can improve both body composition and MOI. Prior investigations have not provided a clear picture; studies have shown that exercise training induces improvements in inflammation independent of body mass index (BMI) [8,9] and others that suggest weight loss-associated reductions in inflammation are independent of exercise [10,11]. The lack of a control group (diet or exercise only) in the majority of these studies has been a major limitation when trying to identify the independent effects of exercise or diet on inflammation [4,6]. On the other hand, few studies have measured inflammatory markers concentrations in adipose tissue [12,13].
The aim of the present study was to investigate the independent and the combined effects of exercise on metabolic factors and inflammation in obese subjects to determine whether exercise training per se, independent of diet, has anti-inflammatory effects. The study was a 24-week randomized intervention with three groups of obese women: (1) exercise alone (EX), (2) hypocaloric diet only (DI), and (3) hypocaloric diet plus exercise (EXD). The inflammatory markers were determined both in the blood circulation and in abdominal fat.

Subjects
Thirty obese healthy females participated in this survey. Obesity was defined as a BMI between 30 and 40 kg m −2 . Exclusion criteria were cardiovascular disease, type 2 diabetes, pregnancy, or orthopedic difficulties causing inability to undertake an exercise program. No subjects received medication that could affect the investigated metabolic markers. Prior to participation, the subjects gave a written informed consent. This study was approved by the local ethics committee of the Faculty of Medicine, University of Sousse, Tunisia. The women gave a written informed consent for the experimental protocol. The 30 obese women were randomized into the 24-week intervention study consisting of (1) EX, (2) hypocaloric DI, or (3) EXD.

Anthropometric Measurements
Height was measured with a standing stadiometer and recorded with a precision of 0.1 cm. Waist circumference was taken as the smallest circumference between the lower costal margin and the pelvic brim measured to the nearest 0.5 cm. Body mass (measured to the nearest 0.1 kg) and percent body fat were measured using bioelectrical impedance analysis (BEURER, Germany). Participants were nude or wearing only underwear for measurements of body mass. BMI was calculated using the standard formula: body mass in kilograms divided by height in meters squared (kg m −2 ). Their physical characteristics are shown in Table 1.

Exercise Stress Test
Participants performed an incremental exercise test to exhaustion on a calibrated cycle ergometer (Ergoline, Germany) to determine maximal oxygen consumption (VO 2max ). The test consisted of a 5-min warm-up followed by increments in power of 1 min at 60 rpm until exhaustion. The loads during warm-up and increments were individually adjusted by taking into account the age, height, and body mass of each subject [14]. The analyzer was calibrated before the rest with the gasses of known concentration. Validation of attainment of VO 2max satisfied two of the following four criteria: (1) an oxygen uptake plateau despite increasing exercise intensity, (2) respiratory exchange ratio ≥1.10, (3) maximal heart rate within 10 beats per minute of the age-predicted maximal values [15], and (4) subject exhaustion.

Exercise Intervention
Aerobic training included three sessions per week of walking/running on a treadmill, starting at 55 % of maximal heart rate for 30 min for weeks 1-4 for 30 min. Exercise intensity and duration were gradually increased every month until subjects exercised at 80 % of maximum heart rate for 45 min at weeks 20-24.

Dietary Protocol
A balanced and personalized dietary restriction program was established by a dietician after an initial dietary assessment in order to define the total amount of calories consumed per day. Subjects in the DI and EXD groups recorded the times and amounts of food and fluid intake for a week before the beginning of the program. The dietary program was set at −500 kcal/day below the initial dietary records. It was composed of 15 % proteins, 55 % carbohydrates, and 30 % lipids. The women recorded, in a specifically designed notebook, the quantity of food and the time at which it was eaten (four times a week). The foods were selected according to the subject's dietary habits. Power Point presentations, videos, and role play scripts were designed for trainers to use during the educational program. Each individual's diet was designed using a Bilnut 4 Software package (SCDA Nutrsoft, Cerelles, France), a computerized database that calculates the food intake and composition from the National Institute of Statistics of Tunis 1978. The body mass was measured every week to assess the immediate effect of the nutritional modifications.

Diet+Exercise Intervention
The EXD intervention combined the above interventions.

Blood Sampling and Analysis
Blood samples were collected by venepuncture on three occasions: at the beginning of the protocol control value (CV), after 12 weeks (S1), and after 24 weeks (S2). Blood samples were collected in the morning at 7-8A.M. after an overnight fast. For menstrual status, all the participants were menstruating regularly and defined as eumenorrheic; all testing was performed during the follicular phase of the menstrual cycle.
The plasma was separated and frozen at −80°C for later analysis. Fasting plasma glucose concentrations were measured using an automated device (AU2700, Olympus, France). The inter-assay coefficient of variability (CoV) was 1.7 %. Fasting plasma insulin was assayed by an IRMA Insulin kit (Immunotech, France). The intra-and inter-assay CoV were 3.3-4 and 3.7-4.8 %, respectively. Insulin resistance was assessed using the homeostatic model assessment for insulin resistance (HOMA-R). The HOMA-R has been validated in obese women and was computed as follows: HOMA-R [insulinemia (microunit per milliliter) ×glycemia (millimole per liter)]/22.5 [16]. Fasting TNF-α and IL-6 were determined using an ELISA kit (Immunotech A Beckman Coulter Company) CoV intra assay precision was ranged between 1.6 and 10 % and inter-assay ranged between 5.4 and 12.8 % for TNF-α and for IL-6 CoV intra assay precision was ranged between 1.6 and 6.8 % and inter-assay ranged between 7.9 and 14.6 %.

Adipose Tissue Biopsies
At baseline and after 24 weeks, the abdominal tissue (AT) biopsies were obtained from the abdominal subcutaneous AT depot 5-10 cm lateral to the umbilicus. The skin was anesthetized with lidocaine (10 mg/ml) before a small incision was made and 200 mg of AT was removed under sterile conditions using a liposuction needle. Immediately after removal, the AT sample was washed in isotonic NaCl, snap-frozen in liquid nitrogen and kept at −80°C.

STATISTICAL ANALYSES
Data are presented as means±SD. Statistical analysis was performed using SPSS 12.0 for windows (SPSS Inc., Chicago, IL, USA). A Kolmogorov-Smirnov test was used to determine the normality of distribution of endocrine and metabolic measures which were found to be nonparametric. ANOVA with repeated measures was performed: 3 (protocols)×3 (groups)×3 (times). When this analysis revealed significant differences, a paired Student's t test was used to identify significant changes between CV to S1 and S2 and a nonpaired Student's t test was used to locate where significant differences existed between EX, DI, and EXD groups. Correlations between measured parameters were assessed by Spearman's correlation. The level of significance was set at P<0.05.

Changes in Anthropometric Parameters and VO 2max
The anthropometric data of the subjects within each group, at the beginning, after 12 weeks, and at the end of the program (after 24 weeks), are shown in Table 1.
Participants in the EXD and EX groups increased their VO 2max with 21 and 18 %, respectively, after 12 weeks (P<0.05) and with 39 and 41 %, respectively, after 24 weeks (P<0.05). Whereas there was no change in the DI group (Fig. 1).

Changes in Metabolic and Hormonal Parameters
The absolute values of the metabolic and inflammatory parameters are presented in Table 2. Only in the EXD group glucose, insulin, and HOMA-R were reduced after 24 weeks with 12 % (P<0.01), 39 % (P<0.05), and 45 % (P<0.05), respectively, while there were no significant changes in the DI and EX groups ( Table 2). Lipid profile was strongly affected by the three interventions. Total cholesterol and triglycerides were reduced similarly in the three groups after 12 weeks (P<0.05 for all) and after 24 weeks (P<0.01 for all). HDL cholesterol was significantly increased in all groups after 12 and 24 weeks (P<0.05 and P<0.01; Table 2).
In the EXD and DI groups, circulating IL-6 was reduced by 5 % (P<0.05) and 1 % (P>0.05), respectively, after 12 weeks and by 30 % (P<0.01) for both after the 24-week intervention. TNF-α was also decreased in DI and EXD groups by approximately 44-57 % (P<0.01) after 24 weeks. There was no significant changes in the EX group (Fig. 2).

Correlations
Our results indicated only significant correlations between circulating and adipose tissue IL-6 and TNF-α in EXD and DI groups. In these two groups, at baseline and after 24 weeks, circulating TNF-α was significantly related to TNF-α in AT (r=0.810 (P<0.01) and r=0.830 (P<0.01)), respectively. Furthermore, IL-6 concentration in plasma before and after 6 months was correlated with the IL-6 concentration in AT (r=0.846 (P<0.01) and r= 0.846 (P=0.01)).

DISCUSSION
The purpose of this study in obese women was to identify the independent effects of exercise and diet alone or in combination on changes in marker of inflammation in circulation and in adipose tissue. The findings of the present study provide randomized controlled trial evidence that a diet alone or combined with chronic aerobic exercise induced weight and fat mass loss and reduces chronic inflammation in plasma and adipose tissue in obese women. However, we did not find a statistically significant effect of exercise training on body composition and on inflammatory markers. We demonstrate a general decrement in the inflammatory markers in the circulation and in adipose tissue in response to marked fat loss in the diet and diet exercise groups. This may be of clinical interest and add to previous findings where we and others have shown that weight loss in obese subjects is beneficial in improving the low-grade inflammatory state associated with obesity [12,17], but we found no additional effect of exercise on the inflammatory profile. Prior investigations have provided controversial findings; some have investigated the effect of exercise, of diet, or of diet combined with exercise and few have combined the three models [12]. Exercise training studies alone reduces inflammatory markers after 8 months moderate-intensity resistance training in elderly women (−22 % in log 10 TNF-α (P<0.05) and −30 % in log 10 IL-6 (P<0.01)) [18] or after 12 weeks aerobic-resistance exercise in obese subjects (−32.6 in TNF-α; P<0.01) [19]. Others demonstrate no effect of 16-week resistance exercise in healthy men [20] or 12-weeks low-intensity exercise training performed by elderly women [6] on inflammatory markers. Diet studies showed that 12-weeks very-low-energy diet (800 kcal/day for 8 weeks followed by a weight maintenance diet for 4 weeks) decreased by 20 % IL-6 concentrations (P<0.01) and weight by −11.1 % (P<0.05) in obese subjects [12].
This finding supports the hypothesis that heavy resistance or combined aerobic-resistance exercise train-ing for at least 12 weeks are more effective than moderate aerobic training in reducing markets of inflammation. The volume of training exercise prescribed in the studies by Cordova et al. [18] (50-min resistance exercise for three times per week) and Ho et al. [19] (15-min resistance exercise combined with 15min aerobic exercise for five times per week) was greater than in our study. These differences in exercise prescription intensity and duration could explain why we and other studies [12,20] with lower volumes of aerobic or resistance training did not measure any significant changes of IL-6 and TNF-α. Thus, 24 weeks of moderate-intensity aerobic exercise training was not sufficient to elicit decreases in IL-6 and TNF-α in obese women.
To summarize, when taking into account differences in study designs, it appears that when exercise interventions are used independently (without diet), there are significant improvements in inflammation [9,20]. However, when exercise is incorporated with dietary weight loss [10] or exercise training is performed without any significant weight loss [21,22], the exercise effect is reduced or lost completely. So, it appears that the effect of exercise on MOI is primarily due to reductions of fat mass rather than a specific effect of exercise alone.
In this study, we utilized a diet and exercise only or in combination in order to determine whether the addition of exercise or diet would result in greater improvements in MOI and in body composition. Our results suggested that MOI, body weight, and fat mass are decreased only in diet and diet-exercise groups. Thus, we demonstrated that much larger reductions in adipose tissue mass might produce more measurable and sustained responses in circulating and adipose tissue concentrations of inflammatory markers. These results are consistent with previous studies that showed that weight loss through energy restriction reduces concentrations of inflammatory biomarkers in obese subjects [10,12]. Strangely, we failed to demonstrate any significant modifications in body composition after 6 months aerobic training in obese women. Huffman et al. [23] observed that despite a significant reduction of adipose tissue mass, 6-month aerobic exercise training did not alter cytokine concentrations to any significant degree in overweight and mild obese subjects. In addition, aerobic exercise for 12 weeks, which increased aerobic capacity and decreased body weight, was found to have no effects on circulating inflammatory markers in these obese patients [12]. In accordance to our results, Ogawa et al. [6] demonstrated that 12 weeks of resistance exercise training performed by elderly women do not modify MOI (IL-6 and TNF-α) and body composition parameters. These findings indicate that the effect of exercise on inflammatory markers is primarily due to reductions of fat mass rather than a specific effect of exercise alone.
Two hypotheses might be suggested to explain the decrease of inflammatory markers after the diet and the diet and exercise interventions. The first is the decrease of fat mass and the second is the decrease of insulin resistance and glucose concentrations after the EXD protocol. In obesity, hypertrophied adipocytes are largely responsible for the secretion of IL-6 and TNF-α [24]. Thus, the change in body fat mass is likely to have been partially responsible for the reduced plasma IL-6 and TNF-α concentrations. This is a favorable metabolic outcome in that IL-6 has been shown to reduce both mRNA and protein expression of glucose transporter 4 protein, leading to reduced glucose uptake [25,26]. So, attenuation of IL-6 should improve glucose uptake, normalizing plasma glucose concentrations. Circulating TNF-α released from both adipose tissue and mononuclear cells bind to TNF-α receptors, inducing activation of serine kinases that can stimulate transcription of inflammatory genes, leading to an increase in inflammatory protein production within target tissues, including muscle, adipose, and the liver [27,28]. This establishes a positive feed-forward loop that further amplifies inflammation and insulin resistance. In line with these observations, Swaroop et al. [29] found a correlation between plasma TNF-α and insulin resistance and Kelly et al. [30] found a correlation between plasma glucose and TNF-α. These observations indicated the plasma glucose concentrations can modulate the changes in TNF-α [30].
In the present study, there was a similar decrease after 24 weeks training in TNF-α and in IL-6 in circulation and in adipose tissue. We speculate that the rate of IL-6 and TNF-α secretion from adipose tissue into the circulation was unaffected and could explain the same values in circulation. The correlation between both changes in adipose tissue and circulating levels of these inflammatory markers is therefore understandable. Conversely, Leggate et al. [13] demonstrated a decrease in IL-6 in subcutaneous adipose tissue and no significant change in circulation after 2 weeks of high-intensity intermittent training in overweight and obese males. These authors suggested that the rate of IL-6 secretion from adipose tissue into the circulation may have been unaltered and could explain why there was no change in circulating IL-6. Other tissues and cells also contribute to circulating IL-6, with only ∼15-35 % of circulating IL-6 at rest deriving from subcutaneous adipose tissue [31]. Within adipose tissue, only around 4-10 % of IL-6 comes from adipocytes [32]; therefore, it is likely that other immune cells such as macrophages are the main source of IL-6 production [33].
The strengths of this study include robust measures because we have not only measure the level of the concentrations of these cytokines in circulation but also in abdominal adipose tissue. The limitations of this research were that the intensity of exercise training was moderate and the absence of an exercise normal weight group. Based on previous study [18], it is very possible that exercise in this normal weight population by itself may have had positive effects on cytokines. As mentioned previously, the present analysis focused only on IL-6 and TNF-α concentrations, rather than looking at concentrations of specific hormones that can modulate inflammation such us C-reactive protein (CRP), IL-15, IL-18 and adiponectin [9,12]. The current study also fixed exclusively on an obese women group, and thus our findings may not translate to other individuals with ages or phenotypes different from those of participants in the current sample. Additionally, studies on men, normal weight or trained individuals, children, and diabetic subjects would be of interest.
In summary, we report that 24-week chronic aerobic exercise was found to have no effect on circulating and AT inflammatory markers despite an increased VO 2max . Rather important body composition modifications were found to have beneficial effects on circulating and AT inflammatory markers in these obese women. It is suggested that more intensive training may be necessary to affect systemic inflammation and that weight loss has a more profound impact for reducing MOI in obese women than exercise.