|Physical exercise reduces circulating lipopolysaccharide and TLR4 activation and improves insulin signaling in tissues of DIO rats.|
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|PMID: 21282367 Owner: NLM Status: MEDLINE|
|OBJECTIVE: Insulin resistance in diet-induced obesity (DIO) is associated with a chronic systemic low-grade inflammation, and Toll-like receptor 4 (TLR4) plays an important role in the link among insulin resistance, inflammation, and obesity. The current study aimed to analyze the effect of exercise on TLR4 expression and activation in obese rats and its consequences on insulin sensitivity and signaling.
RESEARCH DESIGN AND METHODS: The effect of chronic and acute exercise was investigated on insulin sensitivity, insulin signaling, TLR4 activation, c-Jun NH(2)-terminal kinase (JNK) and IκB kinase (IKKβ) activity, and lipopolysaccharide (LPS) serum levels in tissues of DIO rats.
RESULTS: The results showed that chronic exercise reduced TLR4 mRNA and protein expression in liver, muscle, and adipose tissue. However, both acute and chronic exercise blunted TLR4 signaling in these tissues, including a reduction in JNK and IKKβ phosphorylation and IRS-1 serine 307 phosphorylation, and, in parallel, improved insulin-induced IR, IRS-1 tyrosine phosphorylation, and Akt serine phosphorylation, and reduced LPS serum levels.
CONCLUSIONS: Our results show that physical exercise in DIO rats, both acute and chronic, induces an important suppression in the TLR4 signaling pathway in the liver, muscle, and adipose tissue, reduces LPS serum levels, and improves insulin signaling and sensitivity. These data provide considerable progress in our understanding of the molecular events that link physical exercise to an improvement in inflammation and insulin resistance.
|Alexandre G Oliveira; Bruno M Carvalho; Natália Tobar; Eduardo R Ropelle; José R Pauli; Renata A Bagarolli; Dioze Guadagnini; José B C Carvalheira; Mario J A Saad|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2011-01-31|
|Title: Diabetes Volume: 60 ISSN: 1939-327X ISO Abbreviation: Diabetes Publication Date: 2011 Mar|
|Created Date: 2011-03-01 Completed Date: 2011-04-22 Revised Date: 2013-06-30|
Medline Journal Info:
|Nlm Unique ID: 0372763 Medline TA: Diabetes Country: United States|
|Languages: eng Pagination: 784-96 Citation Subset: AIM; IM|
|Department of Internal Medicine, State University of Campinas, Campinas, SP, Brazil.|
|APA/MLA Format Download EndNote Download BibTex|
Analysis of Variance
Enzyme-Linked Immunosorbent Assay
Glucose Clamp Technique
I-kappa B Kinase / metabolism
Insulin / metabolism*
Insulin Resistance / physiology*
Interleukin-6 / metabolism
JNK Mitogen-Activated Protein Kinases / metabolism
Lipopolysaccharides / blood*
Liver / metabolism
Muscle, Skeletal / metabolism
Obesity / metabolism*, physiopathology
Physical Conditioning, Animal / physiology*
Reverse Transcriptase Polymerase Chain Reaction
Toll-Like Receptor 4 / metabolism*
|0/Insulin; 0/Interleukin-6; 0/Lipopolysaccharides; 0/Toll-Like Receptor 4; EC 22.214.171.124/I-kappa B Kinase; EC 126.96.36.199/JNK Mitogen-Activated Protein Kinases|
Journal ID (nlm-ta): Diabetes
Journal ID (hwp): diabetes
Journal ID (pmc): diabetes
Journal ID (publisher-id): Diabetes
Publisher: American Diabetes Association
© 2011 by the American Diabetes Association.
Received Day: 30 Month: 12 Year: 2009
Accepted Day: 13 Month: 11 Year: 2010
Print publication date: Month: 3 Year: 2011
Electronic publication date: Day: 21 Month: 2 Year: 2011
Volume: 60 Issue: 3
First Page: 784 Last Page: 796
PubMed Id: 21282367
Publisher Id: 1907
|Physical Exercise Reduces Circulating Lipopolysaccharide and TLR4 Activation and Improves Insulin Signaling in Tissues of DIO Rats|
|Alexandre G. Oliveira|
|Bruno M. Carvalho|
|Eduardo R. Ropelle|
|José R. Pauli|
|Renata A. Bagarolli|
|José B.C. Carvalheira|
|Mario J.A. Saad|
|Department of Internal Medicine, State University of Campinas, Campinas, SP, Brazil
|Correspondence: Corresponding author: Mario J.A. Saad, firstname.lastname@example.org.
It has become increasingly evident that insulin resistance, induced by obesity, is associated with a chronic systemic low-grade inflammation (1–4). In this context, recent studies from our group and others show that the Toll–like receptor 4 (TLR4) may play a central role in the link among insulin resistance, inflammation, and obesity and that a point mutation in TLR4, which inactivates this receptor, prevents the diet-induced obesity (DIO) activation of IκB kinase (IKKβ) and c-Jun NH2-terminal kinase (JNK), and insulin resistance, suggesting that TLR4 is a key modulator in the cross-talk between inflammatory and metabolic pathways (5–10). TLR4 is an essential receptor for the recognition of lipopolysaccharide (LPS) (11). Moreover, a recent study demonstrated that LPS plasma concentrations increase significantly after the intake of high-fat, high-carbohydrate meals (12), suggesting that this LPS comes from the gastrointestinal tract because LPS is fat-soluble. In addition, it has recently been shown that fat intake leads to increased intestinal permeability for LPS (13).
On the other hand, evidence has emerged that exercise training has anti-inflammatory effects, with minimal side effects, which have been shown to occur in several tissues, including skeletal muscle (14), adipose tissue (15), and probably liver. In rats, exercise training lowers adipose inflammation (16), suggesting that exercise may be a useful therapy. Lifestyle interventions involving exercise clearly improve insulin sensitivity, and possibly inflammation, in obese individuals; yet the mechanisms for these effects are not well understood.
On the basis of data from these studies, we hypothesize that suppression of TLR4 signaling may play an important role in the exercise-induced improvement of insulin sensitivity. Thus, the current study aimed to analyze the effect of exercise on TLR4 expression and activation in obese rats, and its consequences on insulin sensitivity and signaling. We report that DIO induces the expression and activation of TLR4 in muscle, adipose tissue, and liver. Furthermore, both acute and chronic exercise strongly reverse the activation of this pathway and improve insulin signaling, providing a new mechanism by which exercise improves insulin action in obesity and type 2 diabetes. In addition, we show that exercise, both acute and chronic, promotes a reduction in serum LPS in DIO rats.
Male Wistar rats, C3H/HeJ mice, and C3H/HeN mice, their respective controls from the University of Campinas Central Animal Breeding Center, were used in the experiments. All antibodies were from Santa Cruz Technology (Santa Cruz, CA), with the exception of anti-Akt, anti–phospho-Akt, anti–phospho-IKKβ, anti–phospho-IRS-1 Ser307, and anti-TLR4, which were obtained from Cell Signaling Technology (Beverly, MA). TAK-242 (ethyl(6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl] cyclohex-1-ene-1-carboxylate) was synthesized at the Chemistry Institute of University of Campinas. LPS and routine reagents were purchased from Sigma Chemical (St. Louis, MO), unless specified elsewhere.
All experiments were approved by the ethics committee at the State University of Campinas. Eight-week-old male Wistar rats were randomly divided into groups: control (C), fed standard rodent chow and water ad libitum; DIO-sedentary rats (DIO), fed a high-fat diet, as previously used (9), and water ad libitum for 20 weeks; DIO-chronic exercised rats (DIO+CE) and DIO-acute exercised rats (DIO+AE), fed a high-fat diet. Insulin and glucose tolerance tests were performed on these rats after 20 weeks of consumption of the diets and after both the exercise protocols, as described previously (17,18). Because individual rats can vary in their ability to perform swimming, some rats (10–15%) were rejected in the screening for the procedure.
After an overnight fast (∼12 h), a 2-h hyperinsulinemic-euglycemic clamp was conducted in anesthetized catheterized rats with [3-3H]glucose and 2-deoxy-d-[1-14C]glucose to assess glucose metabolism in muscle, as previously described (18–20).
Insulin and interleukin (IL)-6 concentrations were determined by an ELISA (Linco, St. Charles, MO). Serum free fatty acid (FFA) levels were analyzed using NEFA-kit-U (Wako Chemical, Neuss, Germany) with oleic acid as a standard. Glucose was measured from whole venous blood with a glucose monitor (Glucometer; Bayer Diagnostics, New York, NY).
Rats were adapted to swimming for 10 min for 2 days to reduce water-induced stress. Animals swam in groups of three in plastic barrels of 45 cm in diameter that were filled to a depth of 60 cm, and the water temperature was maintained at ∼34°C. The training consisted of daily swimming sessions (1 h/day, 5 days/week, for 8 weeks) with a progressive load increase up to 5% of body weight. These conditions were chosen on the basis of previous studies showing that swimming training with this load improved the physical condition of rats (21). The animals were killed with an overdose of anesthetic (sodium thiopental) at 24 and 36 h after the last session.
Under the same conditions imposed as chronic exercise, the acute exercise protocol consisted of two 3-h bouts, separated by a 45-min rest period, as described previously (18). The animals were killed at 2 and 16 h after this protocol was carried out.
After exercise protocols, rats were anesthetized and used 10–15 min later, i.e., as soon as anesthesia was ensured by the loss of pedal and corneal reflexes, the abdominal cavity was opened, the portal vein was exposed, and 0.2 mL of normal saline was injected with or without insulin (10−6 mol/L). At 30 and 90 s after insulin injection, liver and gastrocnemius and adipose tissue were removed, minced coarsely, and homogenized immediately in extraction buffer, as previously described (22). Extracts were used for immunoprecipitation with MyD88 and Protein A-Sepharose 6MB (Pharmacia, Uppsala, Sweden). The precipitated proteins or whole-tissue extracts were subjected to SDS-PAGE and immunoblotting, as previously described (17,20).
We diluted plasma samples to 20% with endotoxin-free water and then heated them to 70°C for 10 min to inactivate plasma proteins. We then quantified serum LPS with a commercially available Limulus Amebocyte assay from Cambrex (Walkersville, MD) according to the manufacturer’s protocol. We ran samples in duplicate and subtracted the background (23).
The mRNA was determined in the muscle, liver, and adipose tissue using RT-PCR, as previously reported (24). Primer sequences are shown in Supplementary Table 1. Results are expressed as relative expression values, as published previously (1).
Data are expressed as means ± SEM, and the number of independent experiments is indicated. The results of blots are presented as direct comparisons of bands or spots in autoradiographs and quantified by optical densitometry (Scion Image; Scion Corporation, Frederick, MD). For statistical analysis, the groups were compared using a two-way ANOVA with the Bonferroni test for post hoc comparisons. The level of significance adopted was P < 0.05.
Figure 1 shows comparative data regarding the controls, DIO-sedentary rats, and DIO rats submitted to chronic exercise when investigated at 24 h (DIO+CE24h) or 36 h (DIO+CE36h) after the last training session. All DIO animals, submitted to chronic exercise or not, exhibited a higher body weight and epididymal fat than the age-matched C group (Fig. 1A and B). The fasting plasma glucose concentrations were similar among all groups (Fig. 1C). During the glucose tolerance test, glucose and insulin levels were higher in DIO rats, compared with controls, and chronic exercise improved glucose tolerance and reduced insulin levels at all time points (Fig. 1C and D). Serum insulin levels were higher in DIO rats, and chronic exercise was able to reduce this hyperinsulinemia (Fig. 1D). The glucose disappearance rate was lower in the DIO group, and exercise training reversed these alterations (Fig. 1E). A hyperinsulinemic-euglycemic clamp with tracer infusions was also performed to examine the effects of training on glucose metabolism in the skeletal muscle. The glucose infusion rate was lower in the DIO group than in the C group and reestablished to control levels in DIO-exercised rats (Fig. 1F). As shown in Fig. 1G, DIO rats presented a significant reduction in glucose uptake in the skeletal muscle when compared with the control group. In contrast, chronic exercise improved insulin-induced glucose uptake in the muscle of DIO rats (Fig. 1G). Serum levels of FFA were also higher in DIO rats and significantly decreased after swimming training (Fig. 1H).
The TLR4 protein content in muscle, liver, and adipose tissue was higher in the DIO group than in the controls (Fig. 2A–C). Results show that, at 24 h after the last training, there was a marked decrease in TLR4 expression and, after 36 h, this was still decreased when compared with nonexercised obese rats (Fig. 2A–C). Furthermore, the TLR4 mRNA expression was significantly reduced after chronic exercise in muscle, liver, and adipose tissue, compared with DIO rats (Fig. 2D–F).
We next investigated TLR4 pathway activation in two steps: 1) as an early event, TLR4/MyD88 interaction was examined; and 2) for downstream signaling, JNK, IKKβ, and ERK1/2 phosphorylation were studied (25). Skeletal muscle, adipose tissue, and liver of DIO rats exhibited significant increases in the TLR4/MyD88 interaction, compared with the C group (Fig. 2G–I). Conversely, in exercised groups, the TLR4/MyD88 interaction decreased significantly in all tissues studied compared with obese sedentary rats (Fig. 2G–I). IRAK-1, another protein of the TLR4 pathway, showed an increased expression in DIO animals compared with the C group. Conversely, chronic exercise did not have any effect on this protein (Fig. 2J–L).
We also investigated whether this reduction was reflected in IKKβ and JNK phosphorylation, which are downstream of TLR4. As expected, an increase in IKKβ and JNK phosphorylation was found in the muscle, liver, and adipose tissue of DIO rats, compared with controls, and this effect was attenuated in the tissues of chronic-exercised obese rats, compared with sedentary DIO rats (Fig. 3A–F). With regard to ERK1/2, DIO rats exhibited high phosphorylation levels in the three tissues analyzed, compared with control animals (Supplementary Fig. 1). In contrast, we detected a marked reduction in ERK activation after chronic exercise (Supplementary Fig. 1).
We then evaluated the effect of exercise training on an important substrate of these kinases of insulin signaling pathway, namely, IRS-1 Ser307 phosphorylation. This phosphorylation was, on average, markedly upregulated in the muscle, liver, and adipose tissue of obese sedentary rats, compared with controls (Fig. 3G–I). In addition, exercise training was also able to reverse diet-induced IRS-1 Ser307 phosphorylation in the muscle, liver, and adipose tissue of DIO+CE24h and DIO+CE36h rats (Fig. 3G–I).
We then examined the consequences that this exercise-induced improved inflammatory profile exerts on the insulin signaling pathway. In the muscle, liver, and adipose tissue, insulin-induced IRβ, IRS-1 tyrosine phosphorylation, and Akt phosphorylation were reduced by 50–80% in DIO rats, compared with the C group (Fig. 4A–I). On the other hand, insulin-induced IRβ, IRS-1, and Akt phosphorylation were increased in the tissues of DIO+CE24h and DIO+CE36h rats, compared with obese sedentary rats, and approached levels of those found in the C group (Fig. 4A–I). No changes in basal phosphorylation or tissue protein levels were observed among groups (Fig. 4A–I).
Figure 5 shows comparative data for the DIO rats and DIO rats submitted to the acute exercise protocol (DIO+AE2h and DIO+AE16h). No significant variation was found in body weight, epididymal fat, and fasting blood glucose in DIO rats after a single session of exercise, when compared with sedentary obese rats (Fig. 5A–C). Acute exercise improved glucose tolerance and reduced serum insulin levels at all time points after the glucose load (Fig. 5D and E). The glucose disappearance rate was restored at both 2 h and 16 h after acute exercise (Fig. 5E). Acute exercise was also capable of restoring the glucose infusion rate during the hyperinsulinemic-euglycemic clamp (Fig. 5F), accompanied by a significant increase in glucose uptake in the skeletal muscle, compared with DIO rats (Fig. 5G). As expected, serum levels of FFA and IL-6 were higher in obese rats, and acute exercise induced a marked increase in the levels of this substrate, mainly in the DIO+AE2h group (Fig. 5H and I).
In DIO animals submitted to acute exercise, no changes in TLR4 protein expression were observed in muscle, liver, and adipose tissue (data not shown). On the other hand, the acute exercised animals showed lower TLR4 mRNA only in muscle (Supplementary Fig. 2A–C). In contrast, we observed significant reductions in the TLR4/MyD88 interaction in all studied tissues (Fig. 6A–C).
We also investigated the effect of acute exercise on TLR4 downstream signaling. As expected, significant decreases in IKKβ and JNK phosphorylation were found in the muscle, liver, and adipose tissue of acute exercised rats, compared with the DIO group (Fig. 6D–I). The same behavior was found for ERK1/2 phosphorylation after the acute exercise (Supplementary Fig. 1). Acute exercise was also able to reverse the diet-induced IRS-1 Ser307 phosphorylation in muscle, liver, and adipose tissue of both DIO+AE2h and DIO+AE16h rats (Fig. 6J–L).
Acute exercise, as observed in the chronic group, improved insulin-induced tyrosine phosphorylation of IRβ and IRS-1 in the insulin-sensitive tissues (Fig. 7A–F). With regard to Akt, the acute protocol stimulated an impressive increase in serine phosphorylation of this protein in all tissues studied that was similar to that observed in the C group (Fig. 7G–I).
The tumor necrosis factor (TNF)-α and IL-6 mRNA levels were upregulated in the DIO group and were decreased after both exercise protocols in muscle, liver, and adipose tissue of almost all exercised groups (Supplementary Fig. 1D–I).
Serum LPS levels were significantly higher in obese sedentary rats when compared with control animals. Conversely, in animals of the DIO+AE2h group, acute exercise was able to completely reverse the high levels of LPS, and this was still observed at 16 h after the acute exercise (Fig. 8A). Notably, in chronic-exercised animals there was also a reduction in serum LPS levels, compared with the DIO group (Fig. 8A).
To examine whether the exercised-induced reduction in serum LPS plays an important role in the improvement of inflammatory status and insulin sensitivity, we infused LPS into the peritoneum of obese exercised rats immediately after acute exercise. Exercised animals treated with LPS showed serum levels of LPS similar to the DIO animals and presented significant increases in TLR4/MyD88 interaction, IKKβ, and JNK phosphorylation in the muscle and adipose tissue (DIO+AE2h and DIO+AE16h) (Fig. 8A–G), increase in TNF-α mRNA in muscle and adipose tissue, and increase in IL-6 mRNA only in muscle (Supplementary Fig. 2D–I). A significant decrease in Akt serine phosphorylation levels was also achieved in LPS-treated animals (Fig. 8H and I).
We next investigated whether an inhibitor of TLR4 signaling (TAK-242) could mimic the effect of exercise on the inflammatory pathway and insulin signaling in DIO rats. The administration of TAK-242 for 3 days improved inflammatory status in the muscle and adipose tissue of DIO rats, as measured by the reduction of TLR4/MyD88 interaction and phosphorylation of IKKβ and JNK, in parallel with an improvement in insulin-induced Akt phosphorylation (Supplementary Fig. 3A). In DIO+AE2h animals treated with this drug, no additive effect was observed in inflammatory parameters or insulin signaling (Supplementary Fig. 3A). In a similar fashion, C3H/HeJ mice fed a high-fat diet did not present insulin resistance, and we did not observe any increase in IKKβ and JNK phosphorylation; furthermore, exercise had no effect on these parameters or on insulin signaling (Supplementary Fig. 3D–F).
Our results show that physical exercise, both acute and chronic, induces an important suppression in the TLR4 signaling pathway as evidenced by the reduction in an early step of this pathway, i.e., TLR4/MyD88 interaction, and in main downstream targets, such as IKKβ and JNK phosphorylation. These alterations are associated with a significant improvement in insulin resistance, in glucose uptake in muscle, and in the insulin signaling pathway. In DIO rats, circulating levels of LPS were increased and acute and chronic exercise reduced the circulating levels of this TLR4 ligand. Taken together, these data suggest that exercise is effective in reducing chronic low-grade inflammation, because it downregulates TLR4 ligand and an important pathway of the inflammatory response.
The increase in TLR4 protein levels in the muscle, liver, and adipose tissue of DIO rats was partially reversed after chronic exercise. This reduction in TLR4, after chronic exercise, supports findings of previous studies, in which the deletion of TLR4 in mice has been reported to prevent high-fat diet–induced insulin resistance (9,26). There was also a decrease in TLR4 activation and a parallel improvement in insulin signaling and sensitivity. Accordingly, a recent study has shown that MyD88-deficient mice are protected from high-fat diet–induced weight gain and impairment of peripheral glucose metabolism induced by palmitate (27). In line with this, our study demonstrated that a point mutation or pharmacologic blocking of TLR4 protects DIO rats from inflammation and insulin resistance, but had no additive or synergic effects to exercise in insulin signaling.
In accordance with a reduction in TLR4 activation, our data demonstrate reductions in IKKβ and JNK activities after chronic exercise in obese animals, which culminates in downregulation of both TNF-α and IL-6 mRNA. This finding is in contrast with previous studies that demonstrated an increase in IL-6 mRNA with exercise (28,29). Nevertheless, these previous studies investigated IL-6 mRNA levels during exercise, in control animals, and with high or moderate intensities, whereas our data were obtained in obese rats, at least 2 h after the exercise and with low-intensity. It is also important to mention that these cytokines, possibly through dysregulation of the TNF-α converting enzyme/tissue inhibitor of metalloproteinase 3 proteolytic system, have been shown to play an important role in subclinical inflammation in obesity/type 2 diabetes insulin resistance (30). It is well established that interventions that inhibit or attenuate IKKβ or JNK activity significantly improve peripheral insulin sensitivity (31). In this context, a previous study revealed that endurance training in obese, diabetic subjects suppresses the activation of the IKK/NFκB pathway, as proven by an increased abundance of IκB-α and IκB-β (32). Moreover, Ropelle et al. (18) have demonstrated that a single session of exercise is able to reverse obese-induced JNK activation and consequently promote increased insulin sensitivity. The suppression induced by exercise in these pathways is important because these are overactivated by obesity and can negatively regulate insulin signaling through IRS-1 Ser307 phosphorylation (33). Moreover, the activation of the mitogen-activated protein kinase (MAPK) signaling pathway is a mechanism that may also increases IRS-1 Ser307 phosphorylation. Our data demonstrate that physical exercise was capable of decreasing ERK1/2 phosphorylation, which may contribute to improving insulin signaling. Previous studies have observed that MAPK activity increases during exhaustive acute exercise (34–36), but the exercise protocol may contribute in explaining these differences; furthermore, the exercise-induced increase in MAPK phosphorylation rapidly decreases on cessation of exercise session and is completely restored to resting levels at 60 min after exercise (37).
Thus, the decrease in TLR4 expression/activation induced by chronic exercise is intriguing and may contribute to explain the amelioration in inflammation status and insulin sensitivity after chronic exercise. These data are in agreement with a previous hypothesis that a physically active lifestyle promotes anti-inflammatory properties (14), as proven in two longitudinal studies showing that regular training may suppress systemic low–grade inflammation (38,39).
Cani et al. (13) recently hypothesized that bacterial LPS, derived from Gram-negative bacteria residing in the gut microbiota, acts as a triggering factor, linking inflammation to high-fat diet–induced diabetes and obesity. They found that high-fat diet feeding resulted in a significant modulation of the dominant bacterial populations within the gut microflora. A reduction in the number of bifidobacteria, Eubacterium rectal-Clostridium coccoides group and Bacteroides, favoring an increase in the Gram-negative to Gram-positive ratio was observed. This modulation of gut microflora was associated with a significant increase in plasma LPS, fat mass, body weight gain, liver hepatic triglyceride accumulation, insulin resistance, and diabetes. Another study has shown that the treatment of rats with polymyxin B, an antibiotic that specifically targets Gram-negative organisms, reduced LPS expression and hepatic steatosis (40). As expected, an impressive increase in LPS serum level in DIO rats was observed. In contrast, for the first time, we showed a significant reduction in serum LPS levels in chronic-exercised obese rats compared with DIO rats. The reason for this reduction in LPS serum levels in chronic exercised obese rats is not known, but we can speculate that a reduction in gut blood flow during exercise, alterations in intestinal barrier, or reduced LPS absorption by a reduced expression of TLR4 in intestinal cells should be considered (41). On the other hand, the decrease in circulating levels of LPS could also play a role in the reduction of TLR4 mRNA and protein expression induced by chronic exercise, because it is well established that an increase in LPS is associated with increases in TLR4 expression (42). During sepsis, with very high levels of LPS, the modulation of TLR4 mRNA and protein expression is tissue-specific (43). Our findings show that the modulation of TLR4 induced by chronic exercise was similar in the three tissues investigated and probably has a transcriptional regulation, because our data showed a parallel decrease in TLR4 mRNA and protein expression. On the other hand, acute exercise modulated TLR4 mRNA only in muscle. It may be speculated that exercise and other factors, in addition to LPS levels, may also be involved in the modulation of TLR4 expression or signaling, such as intracellular lipid levels and reactive oxygen species levels (44–46), which are increased in DIO rats and decreased after exercise. In this context, earlier studies showed that reactive oxygen species levels increase during exercise and may participate in mechanisms that increase glucose uptake during activity (36). As such, this point deserves further exploration.
Modest weight loss, achieved by diet and exercise, can enhance insulin sensitivity and even reverse insulin resistance (47,48). In our study, obese trained animals showed a slight reduction in weight, which puts in doubt whether improvement in inflammation and insulin sensitivity are consequences of exercise per se or just weight loss effects. To elucidate this, obese animals were submitted to acute exercise, which had no effect on body weight or epididymal fat pads. Our data showed that acute exercise reduced TLR4 signaling and downstream kinases, such as IKKβ and JNK, and, consequently, also reduced IRS-1 Ser307 phosphorylation and improved insulin signaling and sensitivity, as well as induced increased muscle glucose uptake. The main difference between acute and chronic exercise was related to TLR4 protein levels, which did not change after an acute bout of exercise. However, the net effect on TLR4 signaling and insulin resistance was similar. Acute exercise also reduced circulating levels of LPS. This reduction in serum levels of LPS, after an acute bout of exercise, may be related to reduced gut blood flow (49,50). Whether reduced LPS levels may contribute to the reduction in TLR4 signaling and insulin sensitivity is not known, but when we infused a low dose of LPS in obese animals after an acute bout of exercise (to increase circulating levels close to those of obese sedentary animals), the reduction in inflammatory pathways and the improvement in insulin signaling and sensitivity were blunted.
In summary, our results show that physical exercise in DIO rats, both acute and chronic, induces an important suppression in TLR4 signaling pathway in the liver, muscle, and adipose tissue, reduces LPS serum levels, and improves insulin signaling and sensitivity. These data provide considerable progress in our understanding of the molecular events that link physical exercise to an improvement in inflammation and insulin resistance.
fn1This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db09-1907/-/DC1.
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de SÃO PAULO, Conselho Nacional de Pesquisa, and INCT---Obesidade e Diabetes.
No potential conflicts of interest relevant to this article were reported.
A.G.O. researched data, contributed to discussion, wrote the article, and reviewed and edited the article. B.M.C. researched data and reviewed and edited the article. N.T. researched data and reviewed and edited the article. E.R.R. reviewed and edited the article. J.R.P. researched data. R.A.B. researched data. D.G. researched data. J.B.C.C. contributed to discussion and reviewed and edited the article. M.J.A.S. contributed to discussion, wrote the article, and reviewed and edited the article.
The authors thank L. Janeri and J. Pinheiro (Department of Internal Medicine, UNICAMP, Campinas, São Paulo) for technical assistance.
|1.||Weisberg SP,McCann D,Desai M,Rosenbaum M,Leibel RL,Ferrante AW Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin InvestYear: 2003;112:1796–180814679176|
|2.||Hotamisligil GS. Inflammation and metabolic disorders. NatureYear: 2006;444:860–86717167474|
|3.||Shoelson SE,Herrero L,Naaz A. Obesity, inflammation, and insulin resistance. GastroenterologyYear: 2007;132:2169–218017498510|
|4.||Schenk S,Saberi M,Olefsky JM. Insulin sensitivity: modulation by nutrients and inflammation. J Clin InvestYear: 2008;118:2992–300218769626|
|5.||Song MJ,Kim KH,Yoon JM,Kim JB. Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes. Biochem Biophys Res CommunYear: 2006;346:739–74516781673|
|6.||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 ChemYear: 2007;282:35279–3529217916553|
|7.||Kopp A,Buechler C,Neumeier M,et al. Innate immunity and adipocyte function: ligand-specific activation of multiple Toll-like receptors modulates cytokine, adipokine, and chemokine secretion in adipocytes. Obesity (Silver Spring)Year: 2009;17:648–65619148127|
|8.||Kim F,Pham M,Luttrell I,et al. Toll-like receptor-4 mediates vascular inflammation and insulin resistance in diet-induced obesity. Circ ResYear: 2007;100:1589–159617478729|
|9.||Tsukumo DM,Carvalho-Filho MA,Carvalheira JB,et al. Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance. DiabetesYear: 2007;56:1986–199817519423|
|10.||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 InvestYear: 2003;112:1821–183014679177|
|11.||Suganami T,Tanimoto-Koyama K,Nishida J,et al. Role of the Toll-like receptor 4/NF-kappaB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages. Arterioscler Thromb Vasc BiolYear: 2007;27:84–9117082484|
|12.||Ghanim H,Abuaysheh S,Sia CL,et al. Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance. Diabetes CareYear: 2009;32:2281–228719755625|
|13.||Cani PD,Bibiloni R,Knauf C,et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. DiabetesYear: 2008;57:1470–148118305141|
|14.||Greiwe JS,Cheng B,Rubin DC,Yarasheski KE,Semenkovich CF. Resistance exercise decreases skeletal muscle tumor necrosis factor alpha in frail elderly humans. FASEB JYear: 2001;15:475–48211156963|
|15.||Bruun JM,Helge JW,Richelsen B,Stallknecht B. Diet and exercise reduce low-grade inflammation and macrophage infiltration in adipose tissue but not in skeletal muscle in severely obese subjects. Am J Physiol Endocrinol MetabYear: 2006;290:E961–E96716352667|
|16.||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 MetabYear: 2009;296:E1164–E117119276393|
|17.||Carvalho-Filho MA,Ueno M,Hirabara SM,et al. S-nitrosation of the insulin receptor, insulin receptor substrate 1, and protein kinase B/Akt: a novel mechanism of insulin resistance. DiabetesYear: 2005;54:959–96715793233|
|18.||Ropelle ER,Pauli JR,Prada PO,et al. Reversal of diet-induced insulin resistance with a single bout of exercise in the rat: the role of PTP1B and IRS-1 serine phosphorylation. J PhysiolYear: 2006;577:997–100717008371|
|19.||Prada P,Okamoto MM,Furukawa LN,Machado UF,Heimann JC,Dolnikoff MS. High- or low-salt diet from weaning to adulthood: effect on insulin sensitivity in Wistar rats. HypertensionYear: 2000;35:424–42910642336|
|20.||Prada PO,Zecchin HG,Gasparetti AL,et al. Western diet modulates insulin signaling, c-Jun N-terminal kinase activity, and insulin receptor substrate-1ser307 phosphorylation in a tissue-specific fashion. EndocrinologyYear: 2005;146:1576–158715591151|
|21.||Luciano E,Carneiro EM,Carvalho CR,et al. Endurance training improves responsiveness to insulin and modulates insulin signal transduction through the phosphatidylinositol 3-kinase/Akt-1 pathway. Eur J EndocrinolYear: 2002;147:149–15712088932|
|22.||Thirone AC,Carvalheira JB,Hirata AE,Velloso LA,Saad MJ. Regulation of Cbl-associated protein/Cbl pathway in muscle and adipose tissues of two animal models of insulin resistance. EndocrinologyYear: 2004;145:281–29314525909|
|23.||Brenchley JM,Price DA,Schacker TW,et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat MedYear: 2006;12:1365–137117115046|
|24.||Handschin C,Chin S,Li P,et al. Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1alpha muscle-specific knock-out animals. J Biol ChemYear: 2007;282:30014–3002117702743|
|25.||Yang H,Young DW,Gusovsky F,Chow JC. Cellular events mediated by lipopolysaccharide-stimulated toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and Elk-1. J Biol ChemYear: 2000;275:20861–2086610877845|
|26.||Shi H,Kokoeva MV,Inouye K,Tzameli I,Yin H,Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin InvestYear: 2006;116:3015–302517053832|
|27.||Kleinridders A,Schenten D,Könner AC,et al. MyD88 signaling in the CNS is required for development of fatty acid-induced leptin resistance and diet-induced obesity. Cell MetabYear: 2009;10:249–25919808018|
|28.||Steensberg A,Fischer CP,Keller C,Møller K,Pedersen BK. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol Endocrinol MetabYear: 2003;285:E433–E43712857678|
|29.||Gollisch KS,Brandauer J,Jessen N,et al. Effects of exercise training on subcutaneous and visceral adipose tissue in normal- and high-fat diet-fed rats. Am J Physiol Endocrinol MetabYear: 2009;297:E495–E50419491293|
|30.||Monroy A,Kamath S,Chavez AO,et al. Impaired regulation of the TNF-alpha converting enzyme/tissue inhibitor of metalloproteinase 3 proteolytic system in skeletal muscle of obese type 2 diabetic patients: a new mechanism of insulin resistance in humans. DiabetologiaYear: 2009;52:2169–218119633828|
|31.||Hundal RS,Petersen KF,Mayerson AB,et al. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin InvestYear: 2002;109:1321–132612021247|
|32.||Sriwijitkamol A,Christ-Roberts C,Berria R,et al. Reduced skeletal muscle inhibitor of kappaB beta content is associated with insulin resistance in subjects with type 2 diabetes: reversal by exercise training. DiabetesYear: 2006;55:760–76716505240|
|33.||Nguyen MT,Satoh H,Favelyukis S,et al. JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. J Biol ChemYear: 2005;280:35361–3537116085647|
|34.||Ryder JW,Fahlman R,Wallberg-Henriksson H,Alessi DR,Krook A,Zierath JR. Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1. J Biol ChemYear: 2000;275:1457–146210625698|
|35.||Roepstorff C,Vistisen B,Donsmark M,et al. Regulation of hormone-sensitive lipase activity and Ser563 and Ser565 phosphorylation in human skeletal muscle during exercise. J PhysiolYear: 2004;560:551–56215308678|
|36.||Chambers MA,Moylan JS,Smith JD,Goodyear LJ,Reid MB. Stretch-stimulated glucose uptake in skeletal muscle is mediated by reactive oxygen species and p38 MAP-kinase. J PhysiolYear: 2009;587:3363–337319403598|
|37.||Widegren U,Jiang XJ,Krook A,et al. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB JYear: 1998;12:1379–13899761781|
|38.||Mattusch F,Dufaux B,Heine O,Mertens I,Rost R. Reduction of the plasma concentration of C-reactive protein following nine months of endurance training. Int J Sports MedYear: 2000;21:21–2410683094|
|39.||Fallon KE,Fallon SK,Boston T. The acute phase response and exercise: court and field sports. Br J Sports MedYear: 2001;35:170–17311375875|
|40.||Pappo I,Becovier H,Berry EM,Freund HR. Polymyxin B reduces cecal flora, TNF production and hepatic steatosis during total parenteral nutrition in the rat. J Surg ResYear: 1991;51:106–1121907698|
|41.||Musch TI,Eklund KE,Hageman KS,Poole DC. Altered regional blood flow responses to submaximal exercise in older rats. J Appl PhysiolYear: 2004;96:81–8812959955|
|42.||Faure E,Thomas L,Xu H,Medvedev A,Equils O,Arditi M. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation. J ImmunolYear: 2001;166:2018–202411160251|
|43.||Matsumura T,Ito A,Takii T,Hayashi H,Onozaki K. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J Interferon Cytokine ResYear: 2000;20:915–92111054280|
|44.||Kwak YS. Effects of training on spleen and peritoneal exudate reactive oxygen species and lymphocyte proliferation by splenocytes at rest and after an acute bout of exercise. J Sports SciYear: 2006;24:973–97816882631|
|45.||Zanchi NE,Bechara LR,Tanaka LY,Debbas V,Bartholomeu T,Ramires PR. Moderate exercise training decreases aortic superoxide production in myocardial infarcted rats. Eur J Appl PhysiolYear: 2008;104:1045–105218762968|
|46.||Lima FD,Oliveira MS,Furian AF,et al. Adaptation to oxidative challenge induced by chronic physical exercise prevents Na+,K+-ATPase activity inhibition after traumatic brain injury. Brain ResYear: 2009;1279:147–15519422810|
|47.||Turner N,Bruce CR,Beale SM,et al. Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents. DiabetesYear: 2007;56:2085–209217519422|
|48.||Toledo FG,Menshikova EV,Azuma K,et al. Mitochondrial capacity in skeletal muscle is not stimulated by weight loss despite increases in insulin action and decreases in intramyocellular lipid content. DiabetesYear: 2008;57:987–99418252894|
|49.||Granger DN,Richardson PD,Kvietys PR,Mortillaro NA. Intestinal blood flow. GastroenterologyYear: 1980;78:837–8636101568|
|50.||Qamar MI,Read AE. Effects of exercise on mesenteric blood flow in man. GutYear: 1987;28:583–5873596339|
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