GTT was performed 1 wk before the wounding (week 15 of feeding) on a subset of HFD-fed (n = 10) and chow-fed (n = 5) mice. As expected, HFD-fed mice had an impaired response to glucose injection (Fig. 1A), indicating a higher degree of insulin resistance in these mice (diet × time interaction, F1.9,24.3 = tolerance (HFD AUC, 39,132 ± 2445; chow AUC, 28,422 ± 1244; P = 0.002). There were no differences between HFD-Ex and HFD-Sed mice at the 15-wk baseline GTT measure (data not shown).
On a subset of mice (n = 5 HFD-Ex, n = 5 HFD-Sed), GTT was measured at day 1 after wounding in addition to baseline (Fig. 1B). Although HFD-Ex had slightly higher glucose intolerance at baseline (before exercise), there was no significant interaction (activity × time point, F1,8 = 0.160, P = 0.699), nor were there significant time point (baseline vs. day 1, F1,8 = 3.072, P = 0.188) or activity (Ex vs. Sed, F1,8 = 2.971, P = 0.123) main effects, indicating that the exercise intervention had little effect on insulin resistance.
As in previous studies (27,28), HFD feeding slowed wound healing rate (Fig. 2A). When comparing wound healing kinetics in sedentary HFD-fed and chow-fed mice for 10 d after wounding, there was a significant time × diet interaction (F2.9,39.4 = 3.655, P = 0.021) and significant main effects for both diet (F1,13 = 6.495, P = 0.024) and time (F2.9,39.4 = 59.955, P = 0.000). Post hoc analysis revealed that chow-fed mice had significantly smaller wounds starting at day 3 and continuing to day 10 after wounding (Fig. 2A, P < 0.05). We also tested whether exercise training could speed healing in both chow-fed and HFD-fed mice. There was no additional effect of exercise on wound healing in chow-fed mice (Fig. 2B, time × activity interaction, F3.1,43.1 = 0.690, P = 0.567). In HFD-fed mice, RM-ANOVA indicated a nonsignificant time × activity interaction (Fig. 2C, F3.0,33.4 = 1.669, P = 0.192) but a main effect of activity that trended toward significance (Fig. 2C, F1,11 = 3.267, P = 0.098). It should be noted that chow and HFD mice in Figure 2A are identical with Chow-Sed and HFD-Sed in Figures 2B and C, respectively. We have arranged the figure in this fashion because we have found that display of the figure in a 2 × 2 factorial arrangement obscures some of the important comparisons that we have highlighted here.
However, in our previous study in aged mice (21), the major effect of exercise was seen early (within 5 d) after wounding. A qualitative evaluation of our healing data suggested that this might have been the case in the present study as well, because the differences in wound size between HFD-Ex and HFD-Sed were greater in the early part (e.g., day 1–5) compared with that in the late part (e.g., day 8–10). Because of this, and because our original hypothesis was exercise would alter wound inflammation early after wounding, we analyzed the effect of exercise on healing kinetics early (day 0 through day 5) after wounding. When RM-ANOVA was performed only on days 0–5, analysis revealed a nearly significant time × activity interaction (Fig. 2C, F2.2,24.1 = 3.229, P = 0.053) and a significant main effect of activity (Fig. 2C, F1,11 = 4.856, P = .050). Post hoc analysis revealed significant differences in wound size between HFD-Ex and HFD-Sed mice at day 1, day 4, and day 5 after wounding (Fig. 2C, P < 0.05).
Wound gene and protein expression
Healing kinetics data described previously demonstrated a) that exercise effects healing of only HFD-fed and not chow-fed mice and b) that the major effects of the exercise bout happened early (within 5 d) after the wound was applied. This is similar to previous research in our laboratory using aged mice (21). Early responses to wounding consist primarily of inflammation (15), and obesity is known to impair the resolution of the inflammatory state prolonging the healing process (27,28). Thus, as in our previous study, we focused our analyses on day 1, day 3, and day 5 after wounding and limited our analyses to HFD-fed mice because exercise did not appear to affect wound healing in chow-fed mice in any way.
We measured gene and protein expression of proinflammatory cytokines IL-1β and TNF-α as well as the anti-inflammatory cytokine IL-10. Previous research has demonstrated that obesity increases levels of proinflammatory cytokines and reduces levels of anti-inflammatory cytokines in the wound environment (5,12,31,37). Surprisingly, exercise training did not affect gene expression of any of the cytokine markers measured in this study. There was neither day × activity interaction (F2,59 = 1.114, P = 0.335) nor main effect for activity (F1,59 = 0.042, P = 0.839) or day (F2,59 = 0.449, P = 0.640) for TNF-α gene expression (Fig. 3A). Likewise, there was neither day × activity interaction (F2,59 = 1.058, P = 0.729) nor main effect for activity (F1,59 = 0.022, P = 0.882) or day (F2,59 = 0.290, P = 0.749) for IL-1β gene expression (Fig. 3B). Finally, there was neither day × activity interaction (F2,59 = 0.448, P = 0.641) nor main effect for activity (F1,59 = 0.711, P = 0.139) or day (F2,59 = 1.093, P = 0.342) for IL-10 gene expression (Fig. 3C).
Although gene expression was unaltered, we hypothesized that posttranscriptional processes might alter the expression of inflammatory proteins. Therefore, we tested wound protein levels of IL-1β, TNF-α, and IL-10 via ELISA at the same time points. Similar to the results of the gene expression assays, exercise training had no effect on protein expression of these cytokines. There was no significant day × activity interaction (F2,58 = 0.921, P = 0.404) and no significant main effect for activity (F1,58 = 0.285, P = 0.596) for TNF-α (Fig. 4A), although there was a significant main effect of day (F2,59 = 15.152, P = 0.000). Likewise, there was no significant day × activity interaction (F2,56 = 0.060, P = 0.942) and no significant main effect of activity (F1,56 = 0.379, P = 0.540) for IL-1β (Fig. 4B), although again, there was a significant main effect of day (F2,56 = 9.021, P = 0.000). Finally, there was no significant day × activity interaction (F2,55 = 0.304, P = 0.739) and no significant main effect of either activity (F1,55 = 0.329, P = 0.568) or day (F2,55 = 0.150, P = 0.861) for IL-10 (Fig. 4C).
We additionally hypothesized that exercise might affect influx of inflammatory cells by modulating chemokine expression in the wound tissue. Therefore, we measured gene expression of macrophage chemokine MCP-1 and neutrophil chemokine KC. There was no significant day × activity interaction (F2,57 = 0.470, P = 0.627) and no significant main effect for activity (F1,57 = 0.010, P = 0.920) for MCP-1 (Fig. 5A), although there was a significant main effect of day (F2,57 = 13.039, P = 0.000). Likewise, there was no significant day × activity interaction (F2,59 = 0.312, P = 0.733) and no significant main effect of activity (F1,59 = 0.796, P = 0.376) for KC (Fig. 5B), although again, there was a significant main effect of day (F2,59 = 8.480, P = 0.001).
Because we found no effect of exercise on wound inflammation in obese mice, we hypothesized that exercise might affect other aspects of the early healing response such as hemostasis. Therefore, we tested gene expression of PDGF, a growth factor released upon platelet activation. There was no significant day × activity interaction (F2,59 = 0.091, P = 0.913) and no significant main effect of activity (F1,59 = 0.218, P = 0.642) for PDGF (Fig. 5C). There was a significant main effect of day (F2,59 = 11.859, P = 0.000).
The major finding of this study was that short-term treadmill exercise (3 d before and 5 d after wounding) speeds wound healing rate in obese, HFD-fed female mice. This is in line with previous studies in aged mice (21) and older adults (3), but to the best of our knowledge, this is the first report of an exercise effect on cutaneous wound healing using an obesity model. Obesity is known to impede wound healing (38), and this has been demonstrated in animal models of obesity (32).
Because the effect of exercise was seemingly limited to early (day 0 to day 5) postwounding, we hypothesized that exercise would reduce wound inflammation, which has been previously shown to be excessive in obese mice (5,12,31,37) and has been shown to be ameliorated by exercise in aged mice (21). Surprisingly, unlike our previous study in aged mice, exercise seemed not to affect wound site gene or protein expression of inflammatory cytokines TNF-α and IL-1β or of anti-inflammatory cytokine IL-10, nor wound site gene expression of chemokines MCP-1 and KC. This is to our knowledge the first report of an exercise effect on wound healing that is unrelated to alterations in wound site inflammation. This finding warrants further study.
There are some potential limitations to this study, primary of which is the use of only female mice. This was done to remain consistent with previous studies in aged mice (21) and in restraint-stressed mice (19) because these studies were major sources of inspiration for the study reported here. However, both previous studies used mouse strains different from the C57Bl/6J strain used in this study (Balb/cByJ and SKH-1 mice, respectively). The choice of C57Bl/6J mice was made because of their suitability as a model of DIO (36), and this strain of mouse is commonly used in obesity research. It is possible that exercise induces differential effects on wound tissue in these mouse strains, because previous research showed an exercise effect on healing rate that approached significance in young, lean Balb/cByJ mice (21), whereas we detected no difference in lean C57Bl/6J mice.
In female mice, estrogen plays a major role in protection from obesity-impaired wound healing (18). In the aged mouse study previously performed by our laboratory (21), the mice were postmenopausal, and thus, most of the estrogen effect was removed. However, our mice were much younger and premenopausal; thus, estrogen may be playing a protective role in reducing inflammation and speeding healing. This is partially supported by the finding that 6-wk-old male C57Bl/6J mice heal more slowly than their female counterparts (unpublished data). It is possible that an extension of this study to male mice might demonstrate an exercise effect on healing rate and wound inflammation similar to that seen in postmenopausal female mice in our previous aging study. This is a possibility that needs future study.
Several other potential mechanisms for the exercise effect on wound healing in obese mice require further investigation. Clotting and hemostasis represent the earliest responses to wounding (15), happening generally within 30 min after trauma. Exercise is known to positively influence hemostasis, possibly by increasing activity of coagulation factors as well as by increasing reactivity of platelets (24). Obesity and diabetes are linked to a dysregulated hemostasis through the maintenance of a procoagulant state (11). Both obesity and sedentary behavior have been shown to be risk factors for the development of inflammation and hemostatic imbalances (16). Thus, it is possible that exercise-induced alterations in hemostasis may explain the effects of exercise in HFD-fed mice seen in this study, and this potential mechanism should be evaluated. Although we saw no differences in PDGF in this study, it is likely that our first measure at 24 h after healing was taken too late to capture differences in hemostasis because this process generally occurs during the first 30–60 min of healing. The increase in PDGF expression seen at days 3 and 5 after wounding (compared with day 1) in this study likely reflects increased PDGF production by cells such as wound-associated macrophages rather than PDGF production resulting from platelet activation.
A second area that bears consideration for future research is the role of exercise in promoting myofibroblast migration and wound contraction in obese mice. Myofibroblasts are specialized cells that induce wound site contraction and are important in the healing of rodent tissues (15). Treadmill running has been previously shown to induce migration of myofibroblasts into the patellar tendon of female mice (34), a finding that lends some credence to this hypothesis. Recent evidence indicates that DIO causes a delay in myofibroblast differentiation in the wound site of HFD-fed rats (28). Thus, the effect on myofibroblast migration and differentiation may be an important mechanism by which exercise exerts its prohealing effect in obese, HFD-fed mice.
This study is the first to demonstrate an effect of exercise on cutaneous wound healing in a model of obesity. Although exercise sped healing rate early after wounding, contrary to previous aging studies, this was seemingly unrelated to alterations in wound inflammation. Future research in this area should focus on the role of early events after wounding (hemostasis and myofibroblast activity) as well as the differential roles of mouse strain and sex because these are thought to have a major effect on healing kinetics.
This project was partially supported by an American College of Sports Medicine Foundation Doctoral Student Research Grant and a Midwest American College of Sports Medicine Student Research Project Award to BDP.
The authors would like to acknowledge Dr. K. Todd Keylock, assistant professor at Bowling Green State University, who consulted on experimental design.
The authors declare no conflicts of interest.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
1. Allison DB, Fontaine KR, Manson JE, Stevens J, VanItallie TB. Annual deaths attributable to obesity in the United States. JAMA
. 1999; 282 (16): 1530–8.
2. Dovi JV, He LK, DiPietro LA. Accelerated wound closure in neutrophil-depleted mice. J Leukoc Biol
. 2003; 73 (4): 448–55.
3. Emery CF, Kiecolt-Glaser JK, Glaser R, Malarkey WB, Frid DJ. Exercise accelerates wound healing among healthy older adults: a preliminary investigation. J Gerontol A Biol Sci Med Sci
. 2005; 60 (11): 1432–6.
4. Esposito K, Pontillo A, Di Palo C, et al.. Effect of weight loss and lifestyle changes on vascular inflammatory markers in obese women: a randomized trial. JAMA
. 2003; 289 (14): 1799–804.
5. Ferguson MW, Herrick SE, Spencer MJ, Shaw JE, Boulton AJ, Sloan P. The histology of diabetic foot ulcers. Diabet Med
. 1996; 13 (1 suppl): S30–3.
6. Finkelstein EA, Trogdon JG, Cohen JW, Dietz W. Annual medical spending attributable to obesity: payer- and service-specific estimates. Health Aff (Millwood)
. 2009; 28 (5): w822–31.
7. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity among US adults, 1999–2008. JAMA
. 2010; 303 (3): 235–41.
8. Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and trends in obesity among US adults, 1999–2000. JAMA
. 2002; 288 (14): 1723–7.
9. Ford ES. Body mass index, diabetes, and C-reactive protein among U.S. adults. Diabetes Care
. 1999; 22 (12): 1971–7.
10. Gil A, Maria Aguilera C, Gil-Campos M, Canete R. Altered signalling and gene expression associated with the immune system and the inflammatory response in obesity. Br J Nutr
. 2007; 98 (1 suppl): S121–6.
11. Goldberg RB. Cytokine and cytokine-like inflammation markers, endothelial dysfunction, and imbalanced coagulation in development of diabetes and its complications. J Clin Endocrinol Metab
. 2009; 94 (9): 3171–82.
12. Goren I, Muller E, Schiefelbein D, et al.. Systemic anti-TNFalpha treatment restores diabetes-impaired skin repair in ob/ob mice by inactivation of macrophages. J Invest Dermatol
. 2007; 127 (9): 2259–67.
13. Greenberg JA. Correcting biases in estimates of mortality attributable to obesity. Obesity (Silver Spring)
. 2006; 14 (11): 2071–9.
14. Guarnieri G, Zanetti M, Vinci P, Cattin MR, Pirulli A, Barazzoni R. Metabolic syndrome and chronic kidney disease. J Ren Nutr
. 2010; 20 (5 suppl): S19–23.
15. Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res
. 2010; 89 (3): 219–29.
16. Hamer M, Stamatakis E. The accumulative effects of modifiable risk factors on inflammation and haemostasis. Brain Behav Immun
. 2008; 22 (7): 1041–3.
17. Hillon P, Guiu B, Vincent J, Petit JM. Obesity, type 2 diabetes and risk of digestive cancer. Gastroenterol Clin Biol
. 2010; 34 (10): 529–33.
18. Holcomb VB, Keck VA, Barrett JC, Hong J, Libutti SK, Nunez NP. Obesity impairs wound healing in ovariectomized female mice. In Vivo
. 2009; 23 (4): 515–8.
19. Horan MP, Quan N, Subramanian SV, Strauch AR, Gajendrareddy PK, Marucha PT. Impaired wound contraction and delayed myofibroblast differentiation in restraint-stressed mice. Brain Behav Immun
. 2005; 19 (3): 207–16.
20. Karlsson EA, Beck MA. The burden of obesity on infectious disease. Exp Biol Med (Maywood)
. 2010; 235 (12): 1412–24.
21. Keylock KT, Vieira VJ, Wallig MA, DiPietro LA, Schrementi M, Woods JA. Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice. Am J Physiol Regul Integr Comp Physiol
. 2008; 294 (1): R179–84.
22. Khanna S, Biswas S, Shang Y, et al.. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One
. 2010; 5 (3): e9539.
23. Krabbe KS, Pedersen M, Bruunsgaard H. Inflammatory mediators in the elderly. Exp Gerontol
. 2004; 39 (5): 687–99.
24. Lippi G, Maffulli N. Biological influence of physical exercise on hemostasis. Semin Thromb Hemost
. 2009; 35 (3): 269–76.
25. Mangge H, Almer G, Truschnig-Wilders M, Schmidt A, Gasser R, Fuchs D. Inflammation, adiponectin, obesity and cardiovascular risk. Curr Med Chem
. 2010; 17 (36): 4511–20.
26. Monteiro R, Azevedo I. Chronic inflammation in obesity and the metabolic syndrome. Mediators Inflamm
. 2010; 2010: 289645.
27. Nascimento AP, Costa AM. Overweight induced by high-fat diet delays rat cutaneous wound healing. Br J Nutr
. 2006; 96 (6): 1069–77.
28. Paulino do Nascimento A, Monte-Alto-Costa A. Both obesity-prone and obesity-resistant rats present delayed cutaneous wound healing. Br J Nutr
. 2011; 106 (4): 603–11.
29. Percik R, Stumvoll M. Obesity and cancer. Exp Clin Endocrinol Diabetes
. 2009; 117 (10): 563–6.
30. Pischon T, Hankinson SE, Hotamisligil GS, Rifai N, Rimm EB. Leisure-time physical activity and reduced plasma levels of obesity-related inflammatory markers. Obes Res
. 2003; 11 (9): 1055–64.
31. Rosner K, Ross C, Karlsmark T, Petersen AA, Gottrup F, Vejlsgaard GL. Immunohistochemical characterization of the cutaneous cellular infiltrate in different areas of chronic leg ulcers. APMIS
. 1995; 103 (4): 293–9.
32. Seitz O, Schurmann C, Hermes N, et al.. Wound healing in mice with high-fat diet- or ob gene-induced diabetes-obesity syndromes: a comparative study. Exp Diabetes Res
. 2010; 2010: 476969.
33. Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with diabetes. JAMA
. 2005; 293 (2): 217–28.
34. Szczodry M, Zhang J, Lim C, et al.. Treadmill running exercise results in the presence of numerous myofibroblasts in mouse patellar tendons. J Orthop Res
. 2009; 27 (10): 1373–8.
35. 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 (5): E1164–71.
36. West DB, Boozer CN, Moody DL, Atkinson RL. Dietary obesity in nine inbred mouse strains. Am J Physiol
. 1992; 262 (6 Pt 2): R1025–32.
37. Wetzler C, Kampfer H, Stallmeyer B, Pfeilschifter J, Frank S. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J Invest Dermatol
. 2000; 115 (2): 245–53.
38. Wilson JA, Clark JJ. Obesity: impediment to postsurgical wound healing. Adv Skin Wound Care
. 2004; 17 (8): 426–35.
39. Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase–polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem
. 1999; 270 (1): 41–9.
Keywords:©2012The American College of Sports Medicine
OBESITY; TRAINING; INFLAMMATION; MICE