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Original Paper

Neuroimmunomodulation 2011;18:123–130 DOI: 10.1159/000322053

Habitual Physical Exercise Improves Macrophage IL-6 and TNF- � Deregulated Release in the Obese Zucker Rat Model of the Metabolic Syndrome

Leticia Martín-Cordero   a Juan José García   a María Dolores Hinchado   a Elena Bote   a Rafael Manso   b Eduardo Ortega   a

a   Departamento de Fisiología, Grupo de Investigación de Inmunofisiología, Facultad de Ciencias,Universidad de Extremadura, Badajoz , y b   Centro de Biología Molecular ‘Severo Ochoa’ (CSIC-UAM) yDepartamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid , España

lower in the obese rats. This deregulated balance in the re-lease of IL-6 and TNF- � in the obese rats was clearly im-proved following adherence to the program of habitual ex-ercise, reflected by a decrease in the spontaneous release of IL-6 together with a better regulation between the sponta-neous and LPS-induced release of TNF- � , approaching the behavior of the lean healthy rats. In addition, an acute bout of exercise decreased the spontaneous release of IL-6 and increased the spontaneous release of TNF- � in the seden-tary, but not in the exercise-adapted obese rats. Conclusion: MS involves a deregulation of TNF- � and IL-6 release by non-infiltrated peritoneal macrophages, which is improved by habitual physical activity. Copyright © 2010 S. Karger AG, Basel

Introduction

The metabolic syndrome (MS) is a disorder associated with obesity and involves risk factors for type-II diabetes mellitus and arteriosclerosis. Obesity is also associated with a ‘low grade of inflammation’, a term that includes an atypical increase in the systemic concentration of TNF- � , IL-6, IL-1 � and C-reactive protein (CRP) [1] . Both IL-6 and TNF- � are the main inflammatory mol-

Key Words

Exercise � IL-6 � Inflammation � Macrophages � Metabolic syndrome � Obesity � TNF- �

Abstract

Objectives: The first objective was to evaluate whether the metabolic syndrome (MS) involves deregulation of TNF- � and IL-6 release by non-infiltrated peritoneal macrophages, using obese Zucker rats as the experimental model of MS and lean Zucker rats as a reference for healthy control values. The second purpose was to evaluate in the obese rats the effects of habitual exercise and of a bout of acute exercise on the observed MS-associated deregulation in the release of TNF- � and IL-6 by peritoneal macrophages. Methods: The habitual exercise consisted of treadmill running: 5 days/week for 14 weeks and 35 cm/s for 35 min in the last month. The acute exercise consisted of a single session of 25–35 min at 35 cm/s. The constitutive or lipopolysaccharide (LPS)-in-duced release of TNF- � and IL-6 by cultured (24 h, 5% CO 2 , 100% relative humidity) peritoneal macrophages was deter-mined by ELISA. Results: Macrophages from the obese rats released more IL-6 than those from the lean healthy rats, both spontaneously and after LPS stimulation. However, both spontaneous and LPS-induced release of TNF- � was

Received: July 23, 2010 Accepted after revision: October 13, 2010 Published online: November 30, 2010

Dr. Eduardo Ortega Department of Physiology, Faculty of Sciences University of Extremadura ES–06071 Badajoz (Spain) Tel./Fax +34 924 289 300, E-Mail orincon   @   unex.es

© 2010 S. Karger AG, Basel1021–7401/11/0182–0123$38.00/0

Accessible online at:www.karger.com/nim

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Neuroimmunomodulation 2011;18:123–130124

ecules associated with obesity-related inflammation, and they are involved in MS-associated metabolic disorders [2–5] . IL-6 enhances systemic levels of CRP [1, 6] , a good inflammatory marker, which is usually increased in indi-viduals and animals suffering from MS. In fact, IL-6 (with one third of its concentration coming from adipo-cytes) can also induce the release of CRP by adipose tis-sue, confirming a link between obesity and inflamma-tion [7] . IL-6 has a role in the development of hyperlipid-emia, diabetes and hypertension [8] , being clearly involved in the regulation of metabolism. TNF- � has been in-volved in insulin resistance, and it is overexpressed and overproduced in adipose tissue in both rodent models of obesity and obese humans [5, 9, 10] . Nevertheless, the re-lease of TNF- � by adipose tissue is primarily produced by non-fat cells present in the tissue [11] , and inflamma-tory factors released by macrophages can induce inflam-mation in adipocytes by stimulating the transcription of inflammatory genes [12] . In turn, macrophages infiltrat-ing adipose tissue can also be affected by deregulated ad-ipokine production by adipocytes in MS [13] . The ques-tion now is whether non-infiltrated macrophages also have an MS-associated dysfunction in the release of in-flammatory cytokines. Recent studies conducted in our laboratory showed that peritoneal macrophages from obese Zucker rats (a good experimental animal model of MS) have a deregulated release of IL-1 � , both spontane-ously (in the absence of an antigenic stimulus) and in re-sponse to an antigenic stimulus, such as lipopolysaccha-ride (LPS) [14] . Based on this, and taking into account that TNF- � and IL-6 (together with IL-1 � ) are the main cytokines produced by macrophages, the first objective of the present investigation was to evaluate a possible MS-induced deregulation in the release of TNF- � and IL-6 by peritoneal macrophages.

On the other hand, it is well known that physical in-activity and obesity breed lifestyle-related diseases in-

volving low-grade inflammation [15] . Exercise (with or without caloric restriction) has also frequently been rec-ommended for obese people [16] . Indeed, aerobic and ha-bitual exercise is an accepted therapeutic strategy in the management of MS, since it improves the diabetic status and insulin sensitivity, reducing the risk of cardiovascular diseases [17, 18] . Based on the anti-inflammatory effects of exercise [6, 19] , it can also be used as a means to control low-grade systemic inflammation [4, 19, 20] . After the possible detection of a deregulated function of peritoneal macrophages from obese Zucker rats to produce TNF- � and IL-6 (our first objective), the second objective of the present investigation was to test whether habitual exercise could improve this MS-induced deregulation, as well as to test the effect of a single bout of exercise on both sedentary and previously trained obese animals.

Methods

Animals and Experimental Design Forty male Zucker rats (Harlan, UK) were studied, compris-

ing 32 obese Zucker rats (HsdOla:ZUCKER- Lepr fa , homozygous fa/fa ) as the experimental model for the MS study and 8 lean Zucker rats (heterozygous Fa/fa ) as the healthy controls for refer-ence values ( table 1 ). Obese Zucker rats carry a spontaneous obe-sity-causing mutation in the leptin receptor gene ( fa ; Lepr fa is an autosomal recessive mutation on chromosome 5). They exhibit obesity at 4–5 weeks of age, and they have many metabolic char-acteristics in common with human obesity-associated type-II di-abetes, such as insulin resistance, hyperphagia, hyperlipidemia (developing adipocyte hypertrophy and hyperplasia), hypercho-lesterolemia, muscle atrophy and hyperinsulinemia. At the begin-ning of the experiment, the rats were 2 months old and housed in cages with food (A04 diet, SAFE, France) and water ad libitum in the animal facilities of the Medical Faculty of the Autonomous University of Madrid. Animals were undisturbed until they were 10–12 weeks old. Then, the experimental design was targeted at studying the differences between lean and obese rats, the effects of habitual exercise or training in the obese rats, and the respons-

Table 1. Effect of exercise on the body weight of the Zucker rats

Lean sedentary Obese sedentary Obese sedentaryafter acute exercise

Obese trained Obese trained after acute exercise

Weight, g 421.885.4 681.887.3* 667.8812.2* 601.787.7*, ** 591.7810.5*, **

E ach value represents the mean 8 SD of 8 rats/group immediately before sacrifice.* p < 0.01 vs. lean sedentary rats; ** p < 0.01 vs. obese sedentary rats and obese sedentary rats after acute exercise. Habitual exercise

(obese trained rats, with and without performing a bout of acute exercise) diminished body weight. Acute exercise did not signifi-cantly modify the body weight of the obese Zucker rats.

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es to a single bout of acute exercise performed by obese sedentary or obese trained animals.

The obese Zucker rats (n = 32) were divided into 4 experimen-tal groups: sedentary rats (n = 8), sedentary rats that performed a single bout of acute exercise (n = 8), trained rats (n = 8) and trained rats that performed a single bout of acute exercise (n = 8). All an-imals were killed at approximately 6 months of age. Given that rats are animals with nocturnal activity, the light/dark cycle was adapted for the animals to perform exercise during their active phase, with the onset of the dark phase about 2 h before the start of the training sessions (light 1: 30–13: 30 h; dark 13: 30–1: 30 h). The animals were trained habitually between 15: 00 and 18: 00 h. The trained animals were kept at rest for 72 h prior to the collec-tion of biological samples or to the performance of the bout of acute exercise. Following these bouts of acute exercise, peritoneal macrophage extractions were made immediately at the conclu-sion of the session. Sedentary lean Zucker rats [also at the age of 6 months (n = 8)] were used as controls to get reference values in healthy animals. At 08: 30 h on the day the animals were killed, food was withdrawn from all the animals.

Biological samples were taken from anesthetized animals. IL-6 and TNF- � released by peritoneal macrophages were deter-mined in each animal. The comparison of the results between the obese and the lean (non-obese) Zucker rats revealed differences in the inflammatory cytokine release assessed in this experimen-tal model of MS, and the comparison between the different ex-perimental groups of obese animals revealed immunophysiologi-cal adaptations and responses to physical exercise.

Exercise The habitual exercise training consisted of running on a tread-

mill (Li 870, Letica Scientific Instruments, Barcelona, Spain), based on adaptation, progression and maintenance phases in in-tensity and duration from 25 cm/s for 10 min in the first weeks to values close to 35 cm/s for 35 min in the last month. Exercise was performed 5 days per week for 14 weeks. The bout of acute exercise consisted of running on a treadmill for 5 min at 17 cm/s followed by 25–35 min at 35 cm/s without incline.

Ethical Approval The experiment was approved by the Ethical Committees of

the Autonomous University (Madrid, Spain) and the University of Extremadura (Badajoz, Spain) according to the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) and the guidelines of the European Community Council Directives and the Declaration of Helsinki.

Anesthesia and Collection of Biological Samples The fasted animals were anesthetized with isoflurane (AEr-

rane, Baxter, Valencia, Spain), with a starting dose of 2% oxygen and 3.5% isoflurane, and a maintenance dose of 1.5% oxygen and 3% isoflurane.

Peritoneal macrophages were extracted from live animals. The abdominal skin was removed and the peritoneal cavity opened. Then, 30 ml of Iscove medium with 1% penicillin-streptomycin (GIBCO BRL Life Technologies, Gaithersburg, Md., USA) were placed into the peritoneal cavity, the abdomen was massaged, and 20 ml of fluid were removed and deposited in polypropylene tubes. The peritoneal cell suspension (containing macrophages and lymphocytes) was washed (by centrifugation for 10 min at

300 g ). Macrophages and lymphocytes were distinguished mor-phologically under a phase contrast microscope, in accordance with previous assays, using non-specific esterase staining and cy-tometric determination [21] . Macrophages and lymphocytes were then counted separately in a Neubauer chamber, and the perito-neal suspension was adjusted to 1.0 ! 10 6 macrophages/ml in Iscove culture medium complemented with 10% fetal bovine se-rum, 1% penicillin-streptomycin and 1% L -glutamine (GIBCO).

Determination of IL-6 and TNF- � Release by Peritoneal Macrophages 294 � l/well of adjusted macrophage suspension were placed

into sterile 48-well flat-bottom culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, N.J., USA) and incubated (37   °   C in 95% air + 5% CO 2 and 100% relative humidity) with6 � l/well of LPS (50 � g/ml) or 6 � l/well of Iscove (for the deter-mination of the spontaneous release of cytokines) for 24 h. Im-mediately after incubation, culture supernatant was collected, centrifuged (300 g for 5 min) in Eppendorf tubes and then stored at –80   °   C until the time of cytokine assay.

Cytokine levels (IL-6 and TNF- � ) were determined from cul-ture supernatants using commercially available ELISAs [Bio-Source Europe, Nivelles, Belgium (IL-6) and Diaclone, Besançon, France (TNF- � )]. Samples were assayed at optimal concentra-tions and according to the manufacturer’s instructions.

Statistical Analysis Values are given as means 8 SEM. The variables were nor-

mally distributed (tested by the normality test). Student’s t test was used for comparisons between group pairs. The non-para-metric ANOVA, Scheffe F test was used for comparisons among three experimental groups. p ! 0.01 was taken as the minimum significance level.

Results

Peritoneal macrophages from the obese rats released spontaneously more IL-6 (p ! 0.01) than those from the lean rats. The protocol of habitual exercise or training de-creased (p ! 0.01 with respect to the obese sedentary group) the release of this cytokine in the obese rats ( fig. 1 a). Macrophages from the obese rats also released more IL-6 than macrophages from the lean rats in response to LPS (p ! 0.01; fig. 1 b), but the percentage of stimulation was lower (p ! 0.01; fig. 1 c). No significant changes between trained and sedentary obese rats were observed in the LPS-induced release of IL-6, but the percentage of stimu-lation was increased (p ! 0.01 vs. both lean and sedentary obese rats) in the trained animals ( fig. 1 b, c).

In contrast to IL-6, peritoneal macrophages from the obese sedentary rats released spontaneously less TNF- � (p ! 0.01) than those from the lean rats. The protocol of habitual exercise increased (p ! 0.01 vs. the obese seden-tary group) the release of this cytokine in the obese rats

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( fig. 2 a). Peritoneal macrophages from the obese rats also respond worse to LPS than peritoneal macrophages from the lean rats: they have diminished both the LPS-induced release of TNF- � (p ! 0.01; fig. 2 a) and the percentage of stimulation induced by LPS (p ! 0.01; fig. 2 c). The proto-col of the habitual exercise or training program increased (p ! 0.01 vs. the obese sedentary group) the release of TNF- � by macrophages stimulated with LPS, but no sig-nificant changes induced by the exercise were observed in the percentage of stimulation by LPS ( fig. 2 b, c).

A single bout of acute exercise decreased (p ! 0.01) the capacity of macrophages from obese sedentary rats to produce IL-6 ( fig. 3 a), and, as a consequence, increased(p ! 0.01) the percentage of stimulation induced by LPS ( fig. 3 b). This effect was not observed in the trained group of obese rats, in which a single bout of acute exercise de-creased (p ! 0.01) uniquely the LPS-induced release of IL-6 ( fig. 3 c)

Finally, figure 4 shows the effect of a single bout of ex-ercise on the release of TNF- � by peritoneal macrophages

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Fig. 1. IL-6 release by peritoneal macrophages from lean seden-tary, obese sedentary and trained obese Zucker rats. a Spontane-ous release (without antigenic stimulation) by peritoneal macro-phages. b Release by LPS-stimulated macrophages. c LPS-induced stimulation (with 100% corresponding to the spontaneous release

in the absence of LPS activation). Each column represents the mean 8 SD of 8 independent experiments (1/rat) performed in duplicate. *  p ! 0.01 vs. lean sedentary rats; * *  p ! 0.01 vs. obese sedentary rats.

Fig. 2. TNF- � release by peritoneal macrophages from lean sed-entary, obese sedentary and trained obese Zucker rats. a Sponta-neous release (without antigenic stimulation) by peritoneal mac-rophages. b Release by LPS-stimulated macrophages. c Stimula-tion induced by LPS (with 100% corresponding to the spontaneous

release in the absence of LPS activation). Each column represents the mean 8 SD of 8 independent experiments (1/rat) performed in duplicate. *  p ! 0.01 vs. lean sedentary rats; * *  p ! 0.01 vs. obese sedentary rats.

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Fig. 3. Effect of a single bout of acute exer-cise performed by both sedentary and trained obese Zucker rats on IL-6 release by peritoneal macrophages. Spontaneous (without antigenic stimulation); and LPS-induced release of IL-6 by peritoneal mac-rophages from the sedentary ( a ) or the trained ( c ) obese Zucker rats. LPS-induced stimulation (with 100% corresponding to the constitutive release in the absence of LPS activation) in macrophages from the sedentary ( b ) or the trained ( d ) obese Zucker rats is also shown. Each column represents the mean 8 SD of 8 indepen-dent experiments (1/rat) performed in du-plicate. *  p ! 0.01 vs. spontaneous release; * *  p ! 0.01 vs. sedentary with acute exer-cise group; * * *  p ! 0.01 vs. values obtained without acute exercise (Student’s t test).

Fig. 4. Effect of a single bout of acute exer-cise performed by both the sedentary and the trained obese Zucker rats on TNF- � release by peritoneal macrophages. Spon-taneous (without antigenic stimulation);0 and LPS-induced release of TNF- � by macrophages from sedentary ( a ) or trained ( c ) Zucker rats. LPS-induced stimulation (with 100% corresponding to the constitu-tive release in the absence of LPS activa-tion) in macrophages from the sedentary ( b ) or the trained ( d ) obese Zucker rats is also shown. Each column represents the mean 8 SD of 8 independent experiments (1/rat) performed in duplicate. *  p ! 0.01 vs. spontaneous release; * * *   p ! 0.01 vs. values obtained without acute exercise (Student’s t test).

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from obese rats. The acute exercise increased both the spontaneous and the LPS-induced release of TNF- � uniquely in the sedentary obese rats (no significant changes were determined after the acute exercise in the trained group of obese rats).

Discussion

The present investigation was conducted to evaluate a possible deregulation in the inflammatory response of peritoneal macrophages in an experimental animal mod-el that develops MS, the obese Zucker rat ( fa/fa ), as well as to test whether exercise improves macrophage func-tion in MS-associated disorders. First, we evaluated dif-ferences between the animals with MS and the controls (lean healthy rats) in the release of IL-6 and TNF- � by peritoneal macrophages. Then, the animals with MS were studied for the effect of a habitual exercise training pro-gram on these inflammatory parameters. Finally, we studied whether the protocol used for the habitual exer-cise training produced adaptations in the inflamma-tory response of peritoneal macrophages following a sin-gle bout of acute exercise. Obese Zucker rats (HdsOla:Zucker-1 lepr fa ) develop obesity at around 4 weeks of age. It is the most commonly used animal model for the study of genetic obesity and human type-II diabetes mellitus [4] .

In a previous study on obese Zucker rats [14] , it was found that both spontaneous release (in the absence of an antigenic stimulus) and LPS-stimulated release of IL-1 � by peritoneal macrophages are impaired in the obese rats, and this MS-induced disorder was improved by habitual exercise, also reflecting that physical activity can improve the macrophage-mediated immune response to pathogen attacks in animals with MS. Based on this, and taking into account that TNF- � and IL-6, together with IL-1 � , are the main inflammatory cytokines produced by mac-rophages, and that they are the most important cytokines involved in MS, the present investigation has been fo-cused on TNF- � and IL-6. In both rodent models of obe-sity and obese humans, TNF- � is overexpressed and overproduced in the adipose tissue [5, 9, 10] , and it is pri-marily produced by non-fat cells present in the tissue [11] , e.g. infiltrated macrophages, which are affected by a mi-lieu of deregulated adipokine production by adipocytes [13] . However, non-infiltrated peritoneal macrophages from the obese rats released less TNF- � than those from the control lean rats, both spontaneously and following LPS stimulation. This may be explained by the very high

release of IL-6 by the macrophages from the obese ani-mals, above all spontaneously (in the absence of an anti-genic stimulus), in agreement with the concept that IL-6 is a cytokine with inhibitory effects on TNF- � release by monocytes [1] , with special importance in pathologies in-volving low-grade inflammation [22] . Although the high spontaneous release of IL-6 by peritoneal macrophages could contribute to the high circulating levels of this cy-tokine reported in MS, with a potential role in the devel-opment of hyperlipidemia, diabetes and hypertension [8] , results from the present investigation clearly suggest that non-infiltrated peritoneal macrophages cannot be a source for the reported high circulating levels of this cy-tokine (with a potential role in insulin resistance) in Zucker rats [23, 24] . Other cells, such as macrophages in-filtrating the adipose tissue, have to be the main source, and further experimental approaches are being conduct-ed in the experimental model of Zucker rats at present. A good balance in the antigen-activated release of these in-flammatory cytokines is also necessary for the immune response to be optimal. In macrophages from the obese rats, the inflammatory response against a bacterial anti-genic stimulus, such as LPS, is clearly impaired, since LPS-induced release of TNF- � was significantly lower than the reference values obtained in the macrophages from the lean rats. In addition, the difference between spontaneous (no LPS added) and LPS-stimulated IL-6 re-lease was unexpectedly low (clearly due to a very high spontaneous release), also reflecting a low flexibility in the antigenic response by macrophages from the obese animals. These MS-associated disorders in the local in-nate and/or inflammatory response of macrophages may immunocompromise individuals suffering from this pa-thology, in agreement with our previous study evaluating the release of IL-1 � in the same experimental model [14] . Metabolically, regular physical activity is recognized as an effective non-pharmacological strategy in the treat-ment of genetic obesity and type-II diabetes, improving dyslipidemia and insulin sensitivity [15, 25–27] . Bearing the inflammatory mechanisms underlying MS in mind [2, 28, 29] , and based on the anti-inflammatory effects of exercise [6, 19] , it has recently also been reported as a means to control low-grade systemic inflammation [20] . Thus, in studies with obese Zucker rats, the beneficial ef-fects of exercise training on obesity and diabetes have been related to anti-inflammatory mechanisms: for ex-ample, improved circulating levels of CRP, adiponectin [3] , IL-6 and TNF- � [4] , as well as increased levels of the anti-inflammatory cytokines IL-4 and IL-10 [2, 28] . This strongly suggests that exercise training reduces systemic

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inflammation in MS, and these inflammatory changes also correlated with improvements in a variety of meta-bolic abnormalities associated with MS. In a previous study in Zucker rats, we found that habitual exercise im-proves the MS-induced elevation in the spontaneous and LPS-induced release of IL-1 � by peritoneal macrophages [14] . In line with this, the MS-induced deregulation in the release of IL-6 and TNF- � observed in the present inves-tigation is also improved after the protocol of habitual exercise performed by the obese rats, which was reflected by a decrease in the spontaneous release of IL-6 com-bined with a better regulation between the spontaneous and LPS-induced release of TNF- � , confirming the ben-efits of the exercise program. The decrease in the sponta-neous release of IL-6 induced by the habitual exercise al-lows a better regulation of the spontaneous release of TNF- � , with values close to those found in the control lean rats.

Acute exercise enhances innate and/or inflammatory immune responses mediated by macrophages in healthy animals [30–33] . A well-controlled and regulated stimu-lation of innate immune mechanisms during acute exer-cise can help to prevent infections, but overstimulation of the inflammatory response (including the components released by macrophages) could also be harmful to people with inflammatory disorders [34–36] . Although a local macrophage-mediated innate and/or inflammatory re-sponse may be a successful defense against pathogen at-tacks, inappropriate release of proinflammatory cyto-kines, such as IL-1 � , IL-6 or TNF- � , after inadequate ex-ercise (carried out without adaptation or training and/or performed by unhealthy individuals) could also exacer-bate inflammatory disorders. After the single bout of acute exercise, peritoneal macrophages from the seden-tary obese Zucker rats decreased spontaneous release of IL-6 together with increased spontaneous release of TNF- � (reaching higher levels than those determined in the control lean rats). Bearing in mind that TNF- � may be involved in insulin resistance in MS [8, 37] , these results suggest that acute exercise performed by non-trained obese rats could induce a non-optimal inflammatory re-sponse of macrophages in the absence of an antigenic stimulus. However, this effect was not observed when the acute exercise was performed by the previously trained animals, showing a habitual exercise-induced adaptation to respond to an acute exercise. Finally, no significant dif-ferences between obese sedentary and obese trained rats were found in the LPS-induced release of TNF- � after a single bout of exercise, suggesting that acute exercise does not immunocompromise the function of peritoneal mac-

rophages. Even the ‘flexibility’ (percentage of stimula-tion) to produce IL-6 was greater in the sedentary ani-mals following the single bout of exercise.

In a previous report on obese Zucker rats, the benefi-cial effects of exercise on a variety of metabolic abnor-malities associated with MS are related to improvements in the circulating levels of IL-6 and TNF- � [4] . Results presented here also suggest that the spontaneous release of IL-6 and TNF- � is deregulated in peritoneal macro-phages from obese Zucker rats, and this dysfunction can be improved by habitual exercise (raising values close to those found in lean control animals). In addition, the in-creased spontaneous release of TNF- � after a single bout of exercise (with higher values than those found in the lean rats) was uniquely observed in the sedentary group, clearly suggesting a good adaptation induced by the training protocol in the obese rats. Therefore, exercise, at the appropriate duration and intensity, seems to be a good tool for regulating the MS-induced dysfunction in the in-flammatory response mediated by peritoneal macro-phages.

Acknowledgments

This work was partially supported by grants DEP2006-56187-C04-03 (Ministry of Science and Innovation), GRU09004 (Junta de Extremadura), FEDER, RETICEF and Fundacion Val-hondo de Extremadura, Spain. We thank the specialized person-nel from the animal facilities at the Faculty of Medicine of the Autonomous University of Madrid for their collaboration.

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