Low-grade chronic inflammation is attenuated by exercise in obese adults through down-regulation of ASC gene: a pilot study


 Background

Several factors are related to lifestyle behaviors, like physical inactivity and unbalanced diets, which increase the risk of developing obesity and thus represent an important health problem worldwide. Obesity is characterized by low-grade chronic inflammation and an excess of adipose tissue. The ASC protein is part of the NLRP3 inflammasome, a cytosolic multiprotein complex that is associated with inflammation and metabolic alterations.
Purpose

To evaluate the effect of a moderate-intensity structured exercise intervention on ASC gene expression and inflammatory markers in obese adults.
Methods

Thirty-seven obese individuals aged 25 to 50 years were randomized to the diet-exercise group or diet-group. The participants underwent a 4-month follow-up. Electrical bioimpedance was used for body composition analysis. Biochemical data were analyzed by dry chemistry and insulin levels by ELISA. Gene expression from peripheral blood was performed using real-time PCR. Dietary data was collected through questionnaires and analyzed using the Nutritionist Pro™ software. Quantification of cytokines was conducted using Bio-Plex Pro™ Human cytokine. The Astrand-Ryhming test was used to estimate the maximum oxygen volume and design the moderate-intensity structured exercise program.
Results

After the intervention, both study groups significantly improved body composition (decrease weight, fat mass, waist circumference and abdominal obesity, p < 0.05). Besides, the diet-exercise group significantly decreased ASC mRNA expression, MCP-1, and MIP-1β inflammatory cytokines compared to the diet-group (p < 0.05). While in the diet- group, MCP-1 and IL-8 exhibited significantly decreased levels (p < 0.05). In the diet-exercise group, a positive correlation between the atherogenic index and waist circumference was found (r = 0.822, p = 0.011), and a negative correlation was observed between the delta of ASC mRNA expression and IL-10 levels at the end of the intervention (r=-0.627, p = 0.019).
Conclusion

Low-grade chronic inflammation was attenuated through individualized exercise prescription and our findings highlight the role of the ASC gene in the inflammation of obese adults.


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Background Obesity is a global public health concern, representing at least 2.8 million deaths every year [1]. The major complications of obesity include chronic diseases like type 2 diabetes, cardiovascular and metabolic alterations, hypertension, nonalcoholic steatohepatitis (NASH), and some types of cancer [2]. Genetic and environmental factors related to lifestyle have been described to increase adipocyte hypertrophy [3] and the secretion of circulating proin ammatory cytokines [4]. Thus, obesity is associated with different degrees of low-grade chronic in ammation, also known as metabolic in ammation [4], since adipose tissue is related to the de ciency of immune activity and to an ampli ed production of damageassociated molecular patterns (DAMPs) [5,6].
The excess of pro-in ammatory cytokines has been linked with age-related diseases, such as heart failure, metabolic syndrome, insulin resistance, diabetes, arterial hypertension, and asthma [6][7][8].
Particularly, IL-1β and IL-18 pro-in ammatory cytokines mature through the formation and activation of a protein complex, known as NLRP3 in ammasome. The release of DAMPs due to obesity, can activate the NLRP3 in ammasome, and once activated, it recruits a NOD (nucleotide-binding oligomerization domain)-like receptor, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 [9]. The role of ASC protein is to provide stability to the NLRP3 in ammasome. Nonetheless, this protein has been reported as not having in ammatory activities outside of the protein complex. Thereby, it is suggested that the formation and activation of the NLRP3 in ammasome is limited to ASC regulation [10].
It is well known that physical exercise has many health bene ts and it has different molecular responses depending on the frequency, intensity, and type of exercise [11]. Nevertheless, to our knowledge, there is no evidence regarding ASC gene activity in obese adults in response to lifestyle modi cations. Therefore, the aim of this study was to evaluate the effect of moderate-intensity structured exercise intervention on ASC gene expression and in ammatory markers in obese adults.

Baseline characteristics
A total of 61 participants were randomly divided into two groups: the diet-group or the diet-exercise group. However, only 22 participants from the diet-group and 15 participants from the diet-exercise group completed all the study ( Figure 1). Furthermore, age was different between groups, therefore, subsequent statistical analyzes comparing differences between study groups were adjusted for age. The baseline anthropometric and biochemical characteristics are shown in Table 1.

Nutritional intervention
The baseline nutritional analysis did not show differences between groups. However, after the intervention, total energy, total sugar, total lipids, saturated fats, dietary cholesterol, trans fats, and sodium were decreased, and ber, polyunsaturated fatty acids (PUFA) and the consumption of several vitamins were increased. Data are shown in table 2. All physical tests were performed without complications. After the exercise intervention program, no changes in maximum oxygen volume (VO 2max ) and heart rate were observed. However, the watts (W) employed during the test increased signi cantly. These ndings represent a positive adaptation to the exercise program, considering that the signi cant increase in workload (W) does not represent an increased heart rate ( Table 3). Anthropometric and biochemical characteristics after the intervention After four months of the intervention period, both study groups signi cantly improved their body composition; these data are shown in Table 4. The biochemical variables did not show signi cant changes after the intervention, but we observed a discrete tendency in insulin (-4.2 ± 9.2 UI/dL, p=0.09), HOMA-IR (-1.1 ± 2.4, p=0.08) and atherogenic index (-0.3 ± 0.5 p=0.051) only in the diet-exercise group. Then, we compared the body changes between groups after the intervention and we observed a signi cant decrease in musculoskeletal mass (MSM) (p=0.013) and fat-free mass (FFM) (p=0.006) in the control group compared to the diet-exercise group. Also, we observed a signi cant decrease in body fat percentage (BFP) (p=0.029) and atherogenic index (p=0.047) in the diet-exercise group compared to the diet group. In addition, out of the individuals who followed up the diet-exercise program, 26.7% of the subjects decreased abdominal obesity (according to the cutoff point of the International Diabetes Federation) and this change was statistically signi cant compared to the diet group (p=0.026).

ASC mRNA expression
We evaluated the ASC mRNA expression level at baseline time (Time 0) of the intervention and we demonstrated no signi cant differences between the study groups (1±0.204 vs 1.07±0.124, p=0.990) ( Figure 2A). Subsequently, we evaluated the expression levels at Time 4 (four months post-intervention) and we observed that the diet-exercise group signi cantly decreased ASC mRNA expression compared to the diet group (0.485±0.112 vs 1±0.134, p=0.043) ( Figure 2B). Finally, the comparative analysis showed that the diet-exercise group signi cantly decreased ASC mRNA expression compared to baseline time (Time 0) (1±0.116 vs 0.532±0.112, p=0.030) ( Figure 2C). The analysis between groups were adjusted for age, gender, and baseline or nal body fat percentage according to analysis.
The correlation analysis between the variables showed a positive correlation between the atherogenic index and waist circumference within the diet-exercise group (r=0.822, p=0.011) ( Figure 4A). Moreover, in the same study group, a negative correlation was observed between the delta of ASC mRNA expression and IL-10 levels at the end of the intervention (r=-0.627, p=0.019) ( Figure 4B). Additionally, the dietexercise group showed a positive correlation between MIP-1β with abdominal obesity (r=0.642, p=0.033), BFM (r=0.601, p=0.050), BMI (0.657, p=0.028), and BFP (r=0.615, p=0.044), and also between MCP-1 levels with abdominal obesity (r=0.642, p=0.033) and BFP (r=0.769, p=0.015). These correlations were not found in the diet group and all analyses were adjusted for age, gender and nal body fat percentage.

Discussion
Subjects with obesity go through a process known as low-grade chronic in ammation, in turn, this represents a risk factor for the development of metabolic disorders and several diseases, such as nonalcoholic steatohepatitis (NASH) and even cancer. There are different nutritional strategies for the obesity approach, among which, lifestyle changes are the most successful [12]. However, it is important to note that regular exercise has an important anti-in ammatory response. Therefore, we evaluated the effect of a four-month exercise program on ASC mRNA expression and in ammatory markers in obese adults, and we demonstrated that ASC mRNA expression was decreased in obese participants after the diet-exercise program, and a signi cant difference was found compared to the diet-group at the end of the intervention. These results show that exercise has an effect on the expression of the ASC gene.
Other studies have demonstrated hypermethylation of the ASC gene in heart failure patients and in older individuals after exercise, and in turn, methylation was positively correlated with the expression of ASC and IL-1 family, therefore, exercise may play an important role in the epigenetic modi cation of the ASC gene [7,8,13,14]. Although in this study we did not measure IL-1β or IL-18 expression, we quanti ed both cytokines; however, no signi cant differences were found probably because our study population did not have another metabolic disease.
Thus, we propose the ASC gene as a biomarker in response to the exercise intervention to attenuate in ammation in obese individuals and prevent the consequences of low-grade in ammation, since we also observed a negative correlation between the delta of ASC mRNA expression and IL-10 levels in the diet-exercise group.
Interleukin-10 (IL-10) is an important anti-in ammatory cytokine that is associated with the immune response [15], the inhibition of IL-1α, IL-1β, IL-6, IL-8, TNF-α proin ammatory cytokines [16] and the suppression of the NFk-B signaling pathway [17]. Therefore, our results are of interest since the downregulation of the ASC gene in response to the exercise program could be related to the NFk-B signaling pathway, however, this should be explored in future studies.
Furthermore, we also found MCP-1 and MIP-1β decreased levels after the exercise program, and these cytokines are synthesized by monocytes and macrophages [18,19], the main cells where NLRP3 in ammasome is activated [9]. Both cytokines are related to cardiovascular alterations and atherogenic development and are stimulated by proin ammatory cytokines, such as IL-1β. Therefore, we could expect from our ndings that the exercise program had a role in the decrease of NLRP3 in ammasome activation through ASC down-regulation, MCP-1, and MIP-1β; thus, improving the anti-in ammatory pro le through the IL-10 cytokine.
Moreover, visceral adipose tissue surrounding the internal organs has in ammatory activity since it is associated with a greater number of pro-in ammatory cells in the tissue [20]. Therefore, the decreased levels of MCP-1, MIP-1β and IL-8 cytokines in our study also may be in part due to the abdominal fat loss, since these results showed a positive correlation between them (p<0.05).
In this study, both intervention groups improved body composition, which was consistent with other studies where the effect of hypocaloric diets accompanied or not by three months of an exercise intervention program, decreased the same variable [21,22]. These results are expected and partly explained due to the energy restriction involving the activation of lipolytic metabolic pathways, which increase the use of adipose tissue as stored energy [23]. Besides, if the demand for energy increases, as in exercise, the mobilization of fatty acids from stored adipose tissue also increases [24].
In addition, we observed that the participants who performed the diet-exercise program did not have signi cant changes in musculoskeletal and fat-free mass compared to the diet-group, so this indicates that the weight loss was mainly in fat mass. This nding demonstrates some of the additional bene ts of exercise in weight loss management since adding exercise training to energy restriction results in favorable body composition changes in obese subjects. Our results are in accordance with other authors who reported that diet-exercise interventions for six months preserved lean body mass or skeletal muscle mass compared to only diet interventions in obese adults [25,26].
Abdominal obesity has been associated with systemic in ammation [27] and metabolic risk [28]. However, in our study, we found that 26.7% of the participants who performed exercise decreased abdominal obesity, and this was signi cant when compared to the diet-group. Our result is consistent with other studies, which show that regular exercise reduces visceral fat, independent of weight reduction [29,30]. Moreover, O'Donovan et al. found that overweight subjects with better physical tness had lower visceral fat than the overweight un t group [31]. Our results and the evidence mentioned above highlighted the effect of exercise on abdominal fat loss, suggesting that systemic in ammation decreased regardless of weight loss.
In our study, the diet-exercise group showed a tendency to decrease the atherogenic index at the end of the intervention, and a statistical difference was found when we compared the atherogenic index change between groups. Our nding is similar to other results that show an association of exercise with the atherogenic index of plasma after twelve weeks of aerobic exercise in obese individuals [32], as well as a lower atherogenic index in subjects who performed regular exercise compared to sedentary subjects [33]. Lastly, an inverse association was found between the atherogenic index and physical activity levels [34].
In addition, there is evidence that associates obesity as a precursor of atherosclerosis, particularly central adiposity, which is usually determined by waist circumference [35]. In this sense, we found a positive correlation between the atherogenic index and waist circumference in the diet-exercise group, and it is important to mention that this group had a greater abdominal fat loss. Therefore, our results support the evidence that the loss of abdominal obesity could decrease the risk of atherosclerosis, and subsequently, the risk of cardiovascular diseases. This nding highlights the bene ts of exercise as a cardio-protector factor.
Given our ndings, we believe it is important to consider the measurement of ASC methylation, and the expression of the IL-1 family in obese subjects. Also, there were limitations in our study as small sample size and the high dropout rate in the exercise group, which could be due to the lack of commitment and poor adherence to achieve a healthy habit over a long period of time, as well as the subjective method used to quantify the VO2 max , and accelerometers were not used in each exercise sessions. Future researches are needed to done in the same population to support our results and the mechanism of ASC downregulation through exercise and including more markers related to low-grade chronic in ammation pathway.

Conclusion
Our study demonstrated that the structured moderate-intensity exercise program attenuates low-grade chronic in ammation and highlight the role of the ASC gene in the in ammation of obese adults. This study emphasizes the importance of exercise for weight management in individuals with obesity to protect from cardiovascular disease, type 2 diabetes, and metabolic syndrome.  [36] and subjects without history of medication for at least 1 year. Non-inclusion criteria were pregnant or breastfeeding women, diagnosis of diabetes, cardiovascular disease, and cancer, tobacco and alcohol (consumption ≥ 40 g of alcohol per day for men and ≥ 20 g for women) consumption, and muscle or joint injury. All the participants signed a written informed consent. This study was approved by the Ethics and Biosafety Committee of the Health Sciences Center, University of Guadalajara (Registration number CI-08518) and was carried out according to the Declaration of Helsinki (2013).

Intervention
The intervention consisted a 4-month follow up period. The participants were randomly assigned to the diet and exercise program intervention (diet-exercise group), or only diet program intervention (diet-group).
Also, we de ned the baseline measures as Time 0, and the nal measures (4th month) as Time 4.

Astrand-Ryhming test
The participants who performed the diet-exercise intervention, completed an Astrand-Ryhming submaximal test described by Astrand [37] on RECK MOTOmed viva2 cycle ergometer to estimate the VO 2max before the exercise intervention program (50-75 W for woman and 50-100 W for man), and to design the moderate-intensity structured exercise program based on the heart rate, which was monitored during the test in each individual. Once the individuals completed the training program, the nal Astrand-Ryhming test was performed considering the individuals as trained, therefore, the watts of test were greater according to Astrand protocol (75-100 W for woman and 100-150 W for man).

Exercise program intervention
A personal trainer certi ed by the American College of Sports Medicine designed a progressive threephase moderate-intensity exercise program: 1) conditioning phase: 45 minutes/day, 3 days per week for ve weeks (~ 65%HR), 2) progression phase: 1 hour/day, 4 days a week for eight weeks (~ 70-75%HR), and 3) maintenance phase: 1 hour/day, 5 days per week for three weeks (~ 75%HR). The main exercises consisted of improving aerobic (walking and jogging), speed (functional exercise circuits and short races), and resistance performance using dumbbells (weighing less than 5 kilograms). During all sessions, breathlessness and fatigue were measured with the Borg CR10 grade scale (0-10). The personal trainer supervised three sessions per week within the sports facilities of the University Center to ensure that all exercises were performed with an appropriate technique. For the two unsupervised training days, participants were given the detailed training program to perform in the house or park.

Nutritional intervention
The nutrition service carried out the dietary intervention and follow-up evaluation each month. The 24hour diet record and the three-day dietary food record questionnaire (two days during the week and one day of the weekend) were used to collect the dietary information. The Nutritionist Pro™ software (Axxya Systems, Woodinville, WA) was used to estimate the energy intake and food consumption quanti cation. The nutritional intervention consisted of a 20% total energy expenditure reduction estimated by the Mi in-St.Jeor formula [38] using the current weight, with a nutrient distribution as follows: 50% carbohydrates, 30% lipids, and 20% proteins.

Anthropometric and biochemical measurements
Height and weight were measured after an 8-12 hour fast and with participants wearing light clothes.
Height measurements were taken using a stadiometer (Rochester Clinical Research, New York, USA). Waist circumference was measured using a Lufkin Executive® tape and body composition by electrical bioimpedance (InBody 370, Biospace Co. Seoul, Korea) every month of the intervention period.
Peripheral blood samples were taken after an 8-12 hour fast and immediately centrifuged at 3500 rpm to obtain serum. Biochemical variables were performed using a dry chemistry analyzer (Vitros 250 Analyzer, Ortho-Clinical Diagnostics, Johnson & Johnson Services, Inc., Rochester, NY, USA). Low-density lipoprotein cholesterol (LDL-c) was calculated using the Friedewald formula except when triglycerides levels were higher than 400 mg/dL. The atherogenic index was calculated using the formula [Total cholesterol (mg/dL) / HDL-c (mg/dL)], and for cardiovascular risk, values > 3 in women and > 3.5 in men, were considered. Serum insulin levels were determined using Insulin Model ELISA kit, Catalog: CT-600101A (International Diagnostics, S.A de C.V) following the supplier's instructions. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as described by Matthews [39]. A HOMA-IR value > 2.5 was considered as insulin resistance. The biochemical variables were measured every month during the intervention period.
ASC mRNA expression and cytokine levels The ASC mRNA expression and cytokine levels were measured before the start (Time 0) and at the end of the intervention (Time 4

Statistical analysis
The sample size was determined according to the mean difference formula for clinical trials. To achieve a statistical power of 80% and an alpha of 5%, a sample size of 13 participants in each study group was required. However, considering the predicted loss of participants during the study, more than 13 individuals were included per study group. Shapiro-Wilk test was used to evaluate data normality and the Levene's test to verify the homogeneity of the variables. Quantitative variables are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) when the analysis was adjusted for covariables. Statistical differences between groups were analyzed using the unpaired Student's t-test, and paired Student's t-test or Wilcoxon test to evaluate changes between baseline and nal time depending on the normality of the variables, and we used repeated measures ANOVA to observed differences over time.
Pearson and Spearman correlation coe cients were considered normality dependent. A P-value < 0.05 was considered statistically signi cant. All statistical analyses were performed using the software SPSS v.22.0 (IBM, Chicago, IL).

Declarations
Ethics approval and consent to participate All experimental procedures were revised and approved by the Ethics and Biosafety Committee of the Health Sciences Center, University of Guadalajara (Registration number CI-08518).

Consent for publication
Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This research was supported nancially through grants from CONACyT-Sectoriales 202540.
Authors' contributions EML participated in the design of the study and data analysis; EBC participated in the design of the study and contributed to collection and data analysis; KGB contributed to data collection; AMJ contributed to data collection; GRC contributed to collected and data analysis; IHC participated in data analysis. All authors participated in the manuscript preparation, read and approved the nal manuscript. ASC mRNA expression by group over time. The results are shown as mean ± standard error of the mean.
A) There were no signi cant differences in ASC mRNA expression at Time 0 between groups. B) After four months of intervention, the diet-exercise group signi cantly decreased the ASC mRNA expression compared to control group (*p=0.043). C) At the end of the intervention, subjects who performed exercise decreased ASC mRNA expression compared to Time 0 (**p=0.030). Comparison analyses between groups were adjusted for age, gender and baseline or nal body fat percentage according to analysis.