Obesity is associated with the pathogenesis of chronic inflammatory diseases, such as type 2 diabetes (5), which in turn has been largely attributed to the chronic inflammation in visceral adipose tissue (30). Obesity-related adipose tissue inflammation is accompanied by increased infiltration (4) and phenotypic switching (16) of macrophages. Two subsets of macrophages have been operationally defined: M1 macrophages, which exhibit a proinflammatory activation profile, and M2 macrophages, which assume a more immunosuppressive profile (18). The consumption of a high-fat diet (HFD) induces activation and infiltration of M1 macrophages into adipose tissue (19). The M1 macrophages express CD11c and produce inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Recent studies have shown that T cells also play an essential role in the development of adipose tissue inflammation (22,29). CD8+ T cells produce monocyte chemoattractant proteins (MCPs) and macrophage inflammatory proteins (MIPs), which modulate the infiltration of macrophages in adipose tissue (21). Nishimura et al. (20) reported that treatment of obese mice with CD8-specific antibodies attenuates M1 macrophage infiltration and adipose tissue inflammation.
Exercise has anti-inflammatory effects and may prevent and/or dampen the development of chronic inflammatory disease (8). Recently, we reported that exercise training attenuates hepatic inflammation and fibrosis in diet-induced obese mice (13). Similarly, exercise training is now considered to be a crucial mechanism for reducing adipose tissue inflammation (14). We and others have shown that exercise training decreases the expression of TNF-α and the macrophage marker F4/80 in the adipose tissue of obese mice (14,25), suggesting that exercise training may reduce adipose tissue inflammation by suppressing macrophage infiltration. However, in those studies, the increase in F4/80 gene expression reflected macrophage activation, not infiltration (1). Thus, it was probable that the mechanisms by which exercise training might suppress adipose tissue inflammation and infiltration of inflammatory macrophages remain incompletely understood. Inflammatory macrophages and CD8+ T cells are the most abundant cells in adipose tissue of obese individuals, and they have been shown to play critical roles in the associated adipose tissue inflammation (20). In this study, we tested the hypothesis that exercise training changes the number and subset composition of leukocytes in the visceral adipose tissue. We evaluated the presence of adipose-associated leukocytes by separating the stromal vascular fraction (SVF) cells from adipose tissue after collagenase digestion (16) and by evaluating the presence of macrophages and T-cell subsets using flow cytometry analysis.
Animals, diets, and exercise training protocol
Male C57BL/6 mice (4 wk of age) were purchased from Kiwa Laboratory Animals (Wakayama, Japan). Animals were housed (four per cage; 27 × 17 × 13 cm) in a controlled environment with a 12-h light–12-h dark cycle (lights on at 9:00 a.m.). All animals were cared for in conformance with the policy of the American College of Sports Medicine on research with experimental animals. The experimental procedures followed the Guiding Principles for the Care and Use of Animals of the Waseda University Institutional Animal Care and Use Committee (approval no. 2012-A081). The mice were randomly assigned to four groups that received a normal diet (ND) plus sedentary (n = 8), an ND plus exercise training (n = 8), an HFD plus sedentary (n = 12), and an HFD plus exercise training (n = 12). The HFD comprised 60% of calories from fat, 20% from protein, and 20% from carbohydrate (D12492; Research Diets, New Brunswick, NJ). The mice were fed the HFD from 4 to 20 wk of age. The ND mice were fed a standard chow consisting of 10% of calories from fat, 20% from protein, and 70% from carbohydrate (D12450B; Research Diets). All groups had free access to food and water. The animals were weighed weekly, and food intake per cage (four mice) was measured monthly.
Exercise training was initiated when the mice were 4 wk of age and continued for 16 wk. Before the experiment, the mice were trained treadmill running at 15 min once during acclimation period. The exercise training mice were placed on a motorized treadmill (Natsume, Kyoto, Japan) for 60 min·d−1 (during the light phase), 5 d·wk−1, five times a week. The exercise speed was set at 15 m·min−1 for the first 4 wk and 20 m·min−1 for the remaining 12 wk. The mice were not subjected to electric shock during the treadmill sessions to avoid noxious stress. The nonexercised (sedentary) mice remained in their cages. The exercise-trained and untrained mice (20-wk old) were killed 3 d after the final exercise training session under light anesthesia with the inhalant isoflurane (Abbott, Tokyo, Japan). The adipose tissue and liver were quickly removed, weighed, frozen in liquid nitrogen, and stored at −80°C until analysis.
Isolation of splenocytes
Spleen was removed from mice and pressed through 70-μm cell strainers using a syringe barrel. The cell suspension was centrifuged at 800g for 5 min, the cell pellet was resuspended in 2 mL of red blood cell lysing buffer (Sigma-Aldrich, St. Louis, MO), and the suspension was filtered through a 40-μm nylon mesh. The cells were washed twice with the Stain buffer (BD Pharmingen, Franklin Lakes, NJ) and counted using a Scepter Handheld Automated Cell Counter (Millipore, Long Beach, CA).
Isolation of SVF cells from adipose tissue
SVF cells were isolated from the epididymal adipose tissue as previously described, with some modifications (20). The adipose tissue was weighed, minced with scissors, and added to 10 mL of phosphate-buffered saline containing 1 μg·mL−1 of heparin (Sigma). The suspension was centrifuged at 800g for 5 min, and the floating pieces of adipose tissue were collected and placed in 10 mL of Tyrode’s buffer (137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.33 mM NaH2PO4, 5 mM HEPES, and 5 mM glucose) containing 2 mg·mL−1 collagenase type 2 (Worthington, Lakewood, NJ). The mixture was shaken for 20 min at 37°C, and then DMEM medium (Sigma) containing 10% fetal bovine serum was added to the digested tissue. The sample was filtered through a 70-μm mesh and centrifuged at 800g for 5 min. The pellet containing the SVF cells was resuspended in 2 mL of red blood cell lysis buffer and filtered through a 40-μm nylon mesh. The isolated SVF cells were washed twice with staining buffer and counted.
Flow cytometry analysis
The SVF cells and splenocytes (2.5 × 105 cells per sample) were incubated with Fc-blocker (anti-CD16/CD32; eBioscience, San Diego, CA) for 20 min and then stained with combinations of anti-CD11b PE-Cy7, anti-F4/80 PE-Cy5, anti-CD11c PE, anti-CD3[Latin Small Letter Open E] PE Cy7, anti-CD4 PE, and anti-CD8α PerCP Cy5.5 (all from eBioscience) for 20 min. Flow cytometry was performed using a Guava® EasyCyte™ 6HT (Millipore) and InCyte software (Millipore). A validation of the flow cytometric approach for the identification of total CD11b+ F4/80+ macrophages, CD11c+ macrophages, total CD3[Latin Small Letter Open E]+ T cells, CD8α+ T cells, and CD4+ T cells is shown in Supplementary Figure 1 (Supplemental Digital Content 1, http://links.lww.com/MSS/A261).
Real-time quantitative PCR
We analyzed the mRNA levels in epididymal adipose tissue using our previously described methods (14). Gene expression was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as fold change in mRNA levels compared with expression in the ND control group. The sequences of the primer pairs are shown in Supplementary Table 1 (Supplementary Table 1, Supplemental Digital Content 4, http://links.lww.com/MSS/A264).
A sample of epididymal adipose tissue was transferred to a plastic mold, covered with OCT compound, and snap frozen by immersion in precooled isopentane at −80°C. Hematoxylin and eosin (H&E) staining and F4/80 immunohistochemical staining were performed on frozen sections of the adipose tissue using previously described methods (13). Adipocyte size was determined as described previously (24) and was analyzed on low-power (100×) microscopic fields by using BZ-2 software (Keyence, Osaka, Japan). Calculations of the adipocyte size were performed by two independent observers, who were blinded to the diagnosis, and the average value for each section was calculated. The coefficient of variation for the adipocyte size was 7.4%.
All data are expressed as mean ± SEM. Statistical analyses were performed using the Statistical Package for the Social Sciences (Version 19.0; SPSS Inc., Chicago, IL). The statistical significance of differences in body mass, liver and adipose tissue mass, mRNA expression, histological analysis, and flow cytometric analysis was determined using two-way ANOVA with diet (ND or HFD) and exercise (sedentary or exercise training). If significant interactions were observed in any of the analyses, multiple comparisons were performed using Bonferroni post hoc tests after one-way ANOVA. The alpha level was set at P < 0.05.
Effects of HFD and exercise training on physical parameters
Supplementary Figure 2 (Supplemental Digital Content 2, http://links.lww.com/MSS/A262) shows the body weights of each group over the course of the 16-wk study. No statistically significant diet–exercise interaction or exercise effect on body mass at 16 wk was detected using two-way ANOVA, but a statistically significant effect was found for diet (F1,36 = 342.61, P < 0.01; Table 1). A statistically significant diet–exercise interaction was observed for liver and visceral adipose tissue mass (liver, F1,36 = 17.72, P < 0.01; adipose tissue, F1,36 =18.44, P < 0.01). Post hoc comparisons revealed that the liver mass in the HFD sedentary mice was greater than that in the ND sedentary and the HFD exercise mice (P < 0.01; Table 1). The adipose tissue mass was greater in the HFD exercise mice compared with that in the HFD sedentary mice (P < 0.01; Table 1), but the difference in the ND sedentary mice was not observed in the HFD sedentary mice. Calorie intake, which was calculated from the diet and food consumption, varied as a main effect of diet (F1,26 = 45.52, P < 0.01), but no statistically significant diet–exercise interaction was observed (Table 1).
Effects of HFD and exercise training on adipocyte size, SVF number, and inflammatory cytokine gene expression in adipose tissue
We performed H&E staining of adipose tissue to determine adipocyte size and SVF cell clustering around the adipocytes (Fig. 1A). The average size of the adipocytes varied as a diet–exercise interaction (F1,28 = 7.89, P < 0.05). Post hoc comparisons revealed that no difference in adipocyte size in the ND sedentary mice and the HFD sedentary mice, but the cells were much larger in the HFD exercise mice than that in the HFD sedentary mice (P < 0.01; Fig. 1B). We also quantified adipocyte size distribution and found more small adipocytes (cells of ∼1000-μm2 size) in the HFD sedentary mice than that in the ND sedentary and the HFD exercise mice (Figs. 1C and 1D).
As shown in Figure 1A, there were many more clusters in the visceral adipose tissue of the HFD sedentary compared with the ND sedentary and the HFD exercise mice. The SVF cells in adipose tissue are known to produce inflammatory cytokines, but it is unclear whether exercise training changes the SVF cell content. To complement the histological staining, we also analyzed the number of SVF cells, which showed that the number of SVF cells per gram of epididymal adipose tissue varied as a diet–exercise interaction (F1,36 = 19.81, P < 0.01). In addition, post hoc comparisons revealed that the number of SVF cells in the HFD sedentary mice was higher than that in ND sedentary and the HFD exercise mice (P < 0.01; Fig. 1E).
The expression of the inflammatory cytokines TNF-α and IL-6 in the adipose tissue varied as a diet–exercise interaction (TNF-α, F1,36 = 11.74, P < 0.01; IL-6, F1,36 = 4.58, P < 0.05). Post hoc comparisons revealed that the levels of TNF-α and IL-6 mRNA in the HFD sedentary mice were higher than those in the ND sedentary and the HFD exercise mice (TNF-α, P < 0.01; IL-6, P < 0.05; Fig. 1F).
Effects of HFD and exercise training on infiltration of inflammatory macrophages into the adipose tissue
Macrophages are the major sources of inflammatory cytokines associated with obesity-related adipose tissue inflammation. We next determined whether macrophages were present in the adipose tissue by immunohistochemical staining for the specific marker F4/80. The HFD-fed mice showed increased numbers of macrophage clusters compared with the ND-fed mice, but this was markedly lower in the HFD-fed mice subjected to exercise training (Fig. 2A). To examine this further, we isolated SVF cells from the epididymal adipose tissue and examined the expression of macrophage-specific markers by flow cytometry. We found a diet–exercise interaction with respect to the percentage of macrophages in total SVF cells (F1,36 = 9.11, P < 0.01) and the absolute number of macrophages per gram of adipose tissue (F1,36 = 41.07, P < 0.01). Post hoc comparisons revealed that both the percentage and the number of macrophages were increased in the HFD sedentary mice compared with the ND sedentary but were lower in the HFD exercise mice than that in HFD sedentary mice (Figs. 2B and 2C, respectively, P < 0.01).
Adipose tissue macrophages exist as two distinct subtypes that differ in surface marker expression. Whereas F4/80 and CD11b is expressed by all macrophages, CD11c is expressed only by the M1 subtype. We found a diet–exercise interaction (F1,36 = 11.25, P < 0.01; Fig. 2D) for the percentage of CD11c+ cells in the CD11b+ F4/80+ macrophage population. Similarly, we found a diet–exercise interaction with respect to the percentage of CD11c+ macrophages in the SVF cells (F1,36 = 29.58, P < 0.01; Fig. 2E) and the number of CD11c+ macrophages per gram of adipose tissue (F1,36 = 53.83, P < 0.01; Fig. 2F). Post hoc comparisons revealed that compared with the ND sedentary and the HFD exercise mice, adipose tissue from the HFD sedentary mice contained a higher number of CD11c+ macrophages, a higher percentage of CD11c+ macrophages in the total macrophage population, and a higher percentage of CD11c+ macrophages in the SVF cell population (P < 0.01; Figs. 2D–2F).
Effects of HFD and exercise training on T lymphocyte subsets in adipose tissue and spleen
We next investigated the total number of T cells (CD3+ cells) and the number of CD4+ and CD8+ T cells in the adipose tissue and spleen of the mice. We found a diet–exercise interaction with respect to the percentage of CD3+ T cells in SVF cells (F1,36 = 7.31, P < 0.05; Fig. 3A) and the total number of CD3+ T cells per gram of adipose tissue (F1,36 = 16.57, P < 0.01; Fig. 3B). Post hoc comparisons revealed that both parameters were higher in the HFD sedentary mice compared with the ND sedentary mice but were lower in the HFD exercise mice than that in the HFD sedentary mice (P < 0.01). When T-cell subsets were analyzed, we found that the percentage of CD4+ cells in the total T-cell fraction varied according to diet (F1,36 = 7.27, P < 0.05), but the exercise effects and interactions were not significantly different (Fig. 3C). Furthermore, we observed no significant effects of diet and exercise or a diet–exercise interactions on the percentage of CD4+ cells in the SVF cells (Fig. 3D). In contrast, we found a diet–exercise interaction with respect to the percentage of CD8+ cells in the total T-cell fraction (F1,36 = 5.71, P < 0.05) and in the percentage of CD8+ cells in the SVF cells (F1,36 = 7.01, P < 0.05). Post hoc comparisons revealed that both these parameters were higher in HFD sedentary mice than that in the ND sedentary or the HFD exercise mice (Figs. 3C and D, respectively, P < 0.01). Furthermore, the HFD-fed mice had a higher number of CD4+ (4.4-fold) and CD8+ (14.6-fold) cells in adipose tissue than the ND sedentary mice, and this was reduced by exercise (see Supplementary Fig. 3, Supplemental Digital Content 3, http://links.lww.com/MSS/A263). Further analysis of the T-lymphocyte populations indicated that the total number of T cells and the proportion of CD4+ and CD8+ T cells in the spleen were not significantly affected by diet, exercise, or diet–exercise interactions (Fig. 3E).
Effects of HFD and exercise training on the expression of macrophage chemoattractants in adipose tissue
We next examined the expression of several macrophage chemoattractants in adipose tissue. As shown in Figure 4, we found a diet–exercise interaction for the mRNA levels of MCP-1 (F1,36 = 6.25, P < 0.05), MCP-2 (F1,36 = 5.02, P < 0.05), MIP-1α (F1,36 = 21.57, P < 0.01), and MIP-1β (F1,36 = 10.11, P < 0.01). Post hoc comparisons revealed that MCP-1, MCP-2, MIP-1α, and MIP-1β mRNA levels were higher in the HFD sedentary mice than that in the ND sedentary mice (P < 0.01), but these mRNA levels were lower in the HFD exercise mice than that in the HFD sedentary mice (P < 0.01; Fig. 4). The mRNA levels of interferon-inducible protein 10 (IP-10) and regulated on activation normal T cell expressed and secreted (RANTES) were also affected by diet (IP-10, F1,36 = 4.68, P < 0.05; RANTES, F1,36 = 8.16, P < 0.01), but a diet–exercise interaction was not observed.
Adipose tissue inflammation is causally related to the pathogenesis of several chronic inflammatory diseases, including type 2 diabetes and nonalcoholic steatohepatitis. Exercise is known to exert anti-inflammatory effects (8), and we previously reported that exercise training in diet-induced obese mice not only suppressed adipose tissue and hepatic inflammation but also inhibited development of nonalcoholic steatohepatitis (13,14). However, the mechanisms by which exercise training reduces adipose tissue inflammation remain unclear. In this study, we evaluated the effect of exercise training on HFD-induced adipose inflammation and showed that exercise was associated with changes in the infiltration of inflammatory macrophages and CD8+ T cells.
A previous study has shown that exercise training suppresses fat accumulation and induces weight loss in obese rats (32). In the present study, we found that exercise training decreased liver mass but did not induce body weight loss, as we had observed in our previous study (14). Interestingly, HFD induces body weight gain, but the fat mass and average adipocyte size did not change. Strissel et al. (24) reported that epididymal adipose tissue mass and adipocyte size decreased after long-term feeding of an HFD. In addition, adipocyte death and TNF-α gene expression is enhanced by severe obesity (24). TNF-α is known to reduce adipocyte size by increasing lipolysis and adipocyte death (23,31). In this study, we have found that TNF-α mRNA levels were correlated with the percentage of small adipocytes (cells of ∼1000 μm2 size) rather than with the average adipocyte size (r = 0.612, P < 0.01). Thus, HFD feeding-induced severe inflammation may induce loss of adipose tissue mass by reducing adipocyte size. This mechanism may be a primary factor to elucidate why there is no difference in adipose tissue mass between ND mice and HFD mice. However, we also observed that visceral adipose tissue mass and adipocyte size in the obese mice were greatly increased by exercise training. In contrast, expression levels of TNF-α mRNA in adipose tissue were decreased by exercise training. Similarly, the ratio of small adipocytes also was decreased by exercise training. Unlike the previous study (24), the body weight of HFD-treated mice was more than 50 g, which was the state of severe obesity. Therefore, in obese mice, exercise training is likely to reduce the lipolysis and adipocyte death by inhibiting severe adipose tissue inflammation. In fact, the percentage of large adipocytes (cells of ∼10,000-μm2 size per total adipocyte) were higher in the HFD exercise mice (18.95%) compared with the HFD sedentary mice (4.74%). As a result, HFD-treated exercise training mice may have been an increase in average size of adipocytes and fat mass than ND- and HFD-treated sedentary mice. However, whether exercise training affects adipocyte death and lipolysis remains to be determined.
TNF-α and IL-6 are produced by macrophages that promote adipose tissue inflammation (26). Macrophages are present in the stromal cell fraction of adipose tissue, and HFD consumption increases percentage of macrophages in the SVF (16). In this study, we observed that exercise training attenuates expansion of the SVF in adipose tissue. The number of macrophages per gram of visceral adipose tissue changes in obesity because of an increase in the total number of SVF cells (20). In obesity, the influx of macrophages results in the formation of crown-like structures around the adipocytes (24). Consistent with other recent studies, we observed crown-like structures in the adipose tissue of the HFD sedentary mice, and this was markedly reduced by exercise training. We analyzed the characteristics of the SVF in adipose tissue by flow cytometry and observed that exercise training decreased the number of CD11b+ F4/80+ macrophages as a percentage of total SVF cells and also reduced the number of CD11b+ F4/80+ cells per gram of adipose tissue. Recently, Feng et al. (6) reported that injection of clodronate liposomes into diet-induced obese mice reduced macrophage infiltration into the visceral adipose tissue and suppressed adipose tissue inflammation. Thus, the suppression of macrophage infiltration by exercise training may be associated with a reduction of adipose tissue inflammation in obese mice.
Obesity induces an increase in the CD11c+ inflammatory macrophage population and leads to a change in the ratio of macrophage subtypes in mice and humans (16,27). Patsouris et al. (22) reported that, compared with wild-type obese mice, CD11c-deficient obese mice exhibited reduced infiltration of macrophages and reduced levels of TNF-α and IL-6 mRNA. Therefore, the obesity-induced infiltration of inflammatory macrophages is an important factor in the development of adipose tissue inflammation. Interestingly, we have found that exercise training decreases both the proportion and absolute number of CD11c+ macrophages. These results are consistent with our previous study in which we demonstrated that CD11c gene expression in adipose tissue is reduced by exercise training (14). We also observed that TNF-α mRNA levels in adipose tissue correlated with the number of inflammatory macrophages (r = 0.615, P < 0.01). Therefore, the suppression of inflammatory macrophage infiltration by exercise training may play a key role in the reduction of adipose tissue inflammation.
The mechanism by which exercise training reduces macrophage infiltration into adipose tissue is not yet known. Changes in T-cell composition or chemokine production may affect this process. Rausch et al. (22) reported that T cells and macrophages accumulated in obese adipose tissue after intake of an HFD. In contrast to our observations here that CD8+ T cells were increased by an HFD, the number of CD4+ T cells in adipose tissue is reduced (20,28). Interestingly, Nishimura et al. (20) reported that CD11c+ macrophage infiltration and inflammatory cytokine gene expression in adipose tissue were lower in CD8-deficient obese mice than control obese mice. Therefore, CD8+ T cells may play an essential role in the development of adipose tissue inflammation by inducing infiltration of inflammatory macrophages. The effect of exercise training on T-cell content in adipose tissue had previously been unclear.
We showed that exercise training decreases the total number of CD3+ T cells and the ratio of CD8+ to CD4+ cells by reducing the number of CD8+ T cells, but not CD4+ T cells. Furthermore, the CD8/CD4 T-cell ratio in the HFD-treated mice was greatly decreased by exercise training (HFD sedentary vs HFD exercise: 150% vs 89%). Interestingly, this finding was unique to visceral adipose tissue because the number of T cells and T-cell subsets in the spleen did not change with exercise training. In addition, the exercise-induced decrease in CD8+ T cells in adipose tissue was accompanied by a decrease in TNF-α and IL-6 mRNA levels and the number of CD11c+ inflammatory macrophages, suggesting that the ability of exercise training to suppress adipose tissue inflammation and inflammatory macrophage accumulation might be caused by the reduction in CD8+ T-cell numbers.
CD4+ T cells produce anti-inflammatory cytokines such as IL-4 and IL-10, which inhibit activation and infiltration of macrophages into tissues (17). Recent studies have shown that CD4+ T cells in adipose tissue are phenotypically heterogeneous and include regulatory T cells and Th17 cells, which are also associated with adipose tissue inflammation (7,30). Cipolletta et al. (3) reported that immunosuppressive regulatory T cells are reduced in adipose tissue in obesity. In contrast, Th17 cells are increased, and these cells produce MCP-1, which induces macrophage infiltration and adipose tissue inflammation (2). Further work will be required to determine whether exercise training modulates regulatory T cells and Th17 cell populations.
The contribution of chemokines to obesity-induced macrophage infiltration has become apparent in recent years. The expression of macrophage-specific chemokines such as MCPs, MIP-1, IP-10, and RANTES is up-regulated in visceral adipose tissue in obese humans and mice (9,20). MCP-1-overexpressing mice have increased number of macrophages and enhanced TNF-α gene expression in adipose tissue (11,12). Conversely, Ito et al. (10) showed that obese mice deficient in CCR2, the receptor for MCP-1 and MCP-2, showed reduced inflammatory cell infiltration and TNF-α gene expression in adipose tissue compared with control obese mice. Similarly, Kitade et al. (15) showed that obese mice deficient in CCR5 (the receptor for MIP-1α, MIP-1β, and RANTES) showed reduced inflammatory macrophage infiltration and TNF-α gene expression in adipose tissue compared with control obese mice. Interestingly, Nishimura et al. (20) reported that anti-CD8 antibody treatment reduced the expression of these chemokines in adipose tissue, which suggests that chemokines produced by CD8+ T cells may be of central importance for promoting infiltration of macrophages and adipose tissue inflammation. In this study, we found that levels of MCP-1, MCP-2, MIP-1α, and MIP-1β mRNA were significantly reduced by exercise training, but this was not observed for IP-10 and RANTES, although their expression was increased by the HFD. Collectively, our findings indicate that the suppression of macrophage infiltration by exercise training may be associated with a reduction in the expression of chemokines such as MCP-1, MCP-2, MIP-1α, and MIP-1β in adipose tissue.
In summary, we have demonstrated that exercise training markedly reduces the expression of inflammatory cytokines and attenuates the number of CD11c+ M1 macrophages and CD8+ T cells in visceral adipose tissue. Taken together, our results provide evidence that exercise training plays a critical role in reducing adipose tissue inflammation by regulating the infiltration of inflammatory macrophages and CD8+ T cells.
This work was supported by a grant-in-aid for the Global COE (Centers of Excellence) Program “Sport Sciences for the Promotion of Active Life” and the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant-in-aid for the Japan Society for the Promotion of Science Fellows.
There is no conflict of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
1. Bassaganya-Riera J, Misyak S, Guri AJ, Hontecillas R. PPAR gamma is highly expressed in F4/80(hi) adipose tissue macrophages and dampens adipose-tissue inflammation. Cell Immunol
. 2009; 258: 138–46.
2. Cheng X, Wang J, Xia N, et al. A guanidine-rich regulatory oligodeoxynucleotide improves type-2 diabetes in obese mice by blocking T-cell differentiation. EMBO Mol Med
. 2012; 4: 1112–25.
3. Cipolletta D, Feuerer M, Li A, et al. PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature
. 2012; 486: 549–53.
4. Curat CA, Wegner V, Sengenès C, et al. Macrophages in human visceral adipose tissue: increased accumulation in obesity
and a source of resistin and visfatin. Diabetologia
. 2006; 49: 744–7.
5. Dandona P, Aljada A, Bandyopadhyay A. Inflammation: the link between insulin resistance, obesity
and diabetes. Trends Immunol
. 2004; 25: 4–7.
6. Feng B, Jiao P, Nie Y, et al. Clodronate liposomes improve metabolic profile and reduce visceral adipose macrophage content in diet-induced obese mice. PLoS One
. 2011; 6: e24358.
7. Feuerer M, Herrero L, Cipolletta D, et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med
. 2009; 15: 930–9.
8. Gleeson M, Bishop NC, Stensel DJ, Lindley MR, Mastana SS, Nimmo MA. The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease. Nat Rev Immunol
. 2011; 11: 607–15.
9. Huber J, Kiefer FW, Zeyda M, et al. CC chemokine and CC chemokine receptor profiles in visceral and subcutaneous adipose tissue are altered in human obesity
. J Clin Endocrinol Metab
. 2008; 93: 3215–21.
10. Ito A, Suganami T, Yamauchi A, et al. Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue. J Biol Chem
. 2008; 283: 35715–23.
11. Kamei N, Tobe K, Suzuki R, et al. Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance. J Biol Chem
. 2006; 281: 26602–14.
12. Kanda H, Tateya S, Tamori Y, et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity
. J Clin Invest
. 2006; 116: 1494–1505.
13. Kawanishi N, Yano H, Mizokami T, Takahashi M, Oyanagi E, Suzuki K. Exercise training
attenuates hepatic inflammation, fibrosis and macrophage infiltration during diet induced-obesity
in mice. Brain Behav Immun
. 2012; 26: 931–41.
14. Kawanishi N, Yano H, Yokogawa Y, Suzuki K. Exercise training
inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from M1 to M2 macrophages in high-fat-diet-induced obese mice. Exerc Immunol Rev
. 2010; 16: 105–18.
15. Kitade H, Sawamoto K, Nagashimada M, et al. CCR5 plays a critical role in obesity
-induced adipose tissue inflammation and insulin resistance by regulating both macrophage recruitment and M1/M2 status. Diabetes
. 2012; 61: 1680–90.
16. Lumeng CN, Bodzin JL, Saltiel AR. Obesity
induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest
. 2007; 117: 175–84.
17. Miller R, Wen X, Dunford B, Wang X, Suzuki Y. Cytokine production of CD8+ immune T cells but not of CD4+ T cells from Toxoplasma gondii
–infected mice is polarized to a type 1 response following stimulation with tachyzoite-infected macrophages. J Interferon Cytokine Res
. 2006; 26: 787–92.
18. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol
. 2011; 11: 723–37.
19. Nguyen MT, Favelyukis S, Nguyen AK, et al. A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways. J Biol Chem
. 2007; 282: 35279–92.
20. Nishimura S, Manabe I, Nagasaki M, et al. CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity
. Nat Med
. 2009; 15: 914–20.
21. Patsouris D, Li PP, Thapar D, et al. Ablation of CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals. Cell Metab
. 2008; 8: 301–9.
22. Rausch ME, Weisberg S, Vardhana P, Tortoriello DV. Obesity
in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration. Int J Obes
. 2008; 32: 451–63.
23. Ruan H, Hacohen N, Golub TR, Van Parijs L, Lodish HF. Tumor necrosis factor-alpha suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-alpha is obligatory. Diabetes
. 2002; 51: 1319–36.
24. Strissel KJ, Stancheva Z, Miyoshi H, et al. Adipocyte death, adipose tissue remodeling, and obesity
. 2007; 56: 2910–8.
25. Vieira VJ, Valentine RJ, Wilund KR, Antao N, Baynard T, Woods JA. Effects of exercise and low-fat diet on adipose tissue inflammation and metabolic complications in obese mice. Am J Physiol Endocrinol Metab
. 2009; 296: E1164–71.
26. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW. Obesity
is associated with macrophage accumulation in adipose tissue. J Clin Invest
. 2003; 112: 1796–808.
27. Wentworth JM, Naselli G, Brown WA, et al. Pro-inflammatory CD11c+CD206+ adipose tissue macrophages are associated with insulin resistance in human obesity
. 2010; 59: 1648–56.
28. Winer S, Chan Y, Paltser G, et al. Normalization of obesity
-associated insulin resistance through immunotherapy. Nat Med
. 2009; 15: 921–9.
29. Wu H, Ghosh S, Perrard XD, et al. T-cell accumulation and regulated on activation, normal T cell expressed and secreted upregulation in adipose tissue in obesity
. 2007; 115: 1029–38.
30. Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role in the development of obesity
-related insulin resistance. J Clin Invest
. 2003; 112: 1821–30.
31. Xu H, Sethi JK, Hotamisligil GS. Transmembrane tumor necrosis factor (TNF)-alpha inhibits adipocyte differentiation by selectively activating TNF receptor 1. J Biol Chem
. 1999; 274: 26287–895.
32. Yan ZC, Liu DY, Zhang LL, et al. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta. Biochem Biophys Res Commun
. 2007; 354: 427–33.