Contracting skeletal muscle cells play an active endocrine role in the regulation of metabolism and inflammation through production of cytokines in human endurance athletes competing in marathons, ultramarathons, and triathalons. These cytokines might be detected in serum and some have been associated with the exercise-induced acute-phase response (APR). The acute-phase response is a rapid, nonspecific systemic response occurring secondary to many types of tissue injury and might be a physiologic protective mechanism during inflammatory events. The exact cause for the APR, has not been elucidated; although increased serum CRP concentrations occur in endurance racing huskies.[2, 3] A consistent marker for the inflammation of exercise is often observed as increase in macrophage chemoattractant protein-1 (MCP-1).
Three cytokines, now termed myokines, IL-6, IL-8, and IL-15, have been associated with release from contracting skeletal muscle. Of these three, IL-6 is the most extensively studied, whereas less is known about IL-15 and IL-8. An exponential increase in circulating serum concentrations of IL-6 in response to prolonged exercise, followed by a decline in the postexercise period, is a consistent finding in human endurance athletes, and plays a role in acute inflammation in muscle as well as glucose metabolism.[1, 6] IL-15 is an anabolic factor that is highly expressed in skeletal muscle and plays a role in muscle-adipose tissue “cross-talk,” associated with fatty acid oxidation, and decreases with muscle atrophy.[5, 7] Increase in IL-15 serum concentration during exercise might be because of an increased need for fatty acid oxidation and metabolism in contracting skeletal muscle. IL-15 production by type-II muscle fibres is influenced by strength training and resistance exercise with potential for detection of slight increase in serum after exercise. IL-8, a chemokine, is a locally active angiogenic factor in skeletal muscle. Increase in plasma concentrations during exercise response occurs inconsistently during strenuous running exercise in trained and untrained individuals.[5, 10]
C-reactive protein is a marker of systemic inflammation and the APR in both humans and dogs, and IL-6, TNF-α, MCP-1, or all might be associated with this change.[4, 5, 11] The inflammatory response, although mild might be an indication of muscle inflammation and has the potential to be a marker of changes that might be associated with rhabdomyolytic events, as there is an inflammatory change observed in exertional rhabdomyolysis in sled dogs, with invasion of damaged tissue by these inflammatory cells promoting with potential for increased TNF-α, IL-6, and MCP-1 production, which act as chemoattractants.
The potential severe muscle fatigue/injury and pronounced CRP response in sled dogs competing in long-distance races suggest that they might serve as a model for the study of myokines, exercise-induced stress and muscle damage, as these dogs cover greater distances (80–100 miles/day) and are active for longer periods of time than human athletes. The aims of this study were 2-fold: to examine the myokine (IL-6, IL-8, and IL-15) and inflammatory/APR (TNF-α, IL-6, CRP, and MCP-1) responses in trained sled dogs participating in the 2011 Yukon Quest Sled dog race before, midrace, and at the end of the race, hoping to provide insight regarding metabolic and inflammatory changes in muscle associated with strenuous exercise that might one day lead to important biomarkers of exercise stress and muscle damage; and to examine associations between IL-6, MCP-1, and CRP and associations between body weight and IL-15, hoping to divulge the potential mechanisms for the robust CRP response in exercising sled dogs and to examine if IL-15 might be a marker of lean mass changes in exercising sled dogs that lose weight while racing.
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- Materials and Methods
Of the 26 dogs that completed the race and had adequate serum sample for analysis, 8 were from team 1, 10 were from team 2, and 8 were from team 3. The finishing times of teams 1, 2, and 3, respectively, were 10 days, 14 hours, 45 minutes; 12 days, 7 hours, 15 minutes; and 13 days, 2 hours, 26 minutes. Physical examination of each of these dogs at the corresponding check points showed no illness or injury associated with racing. Data from the 28 dogs that completed the race showed a mean weight at the start of 23.2 ± 3.5 kg, which remained similar at the midpoint (24.0 ± 3.3 kg) and then decreased significantly between the midpoint and the finish (22.8 ± 3.5; P = .012). The median serum CRP concentration increased significantly from the start (18 ug/mL) to the midpoint (76 μg/mL), and remained elevated at the finish (60 μg/mL; P < .001). There was no significant difference in median serum CRP concentration between the midpoint and finish (Fig 1A).
Figure 1. Box and whisker plots depicting the median serum CRP (A), MCP-1 (B), IL-6 (C), IL-15 (D), IL-8 (E), and TNF-α (F) from 26 sled dogs before the race start, at the midpoint, and at the finish of a long-distance endurance race. Box represents the 75th and 25th percentiles while whiskers extend to the 95th and 5th percentiles. Outliers are depicted as open circles beyond the 95th and 5th percentiles. * indicates a significant difference from race start (resting) values.
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The median serum MCP-1 concentration increased significantly from the start (85.9 pg/mL) to the midpoint (179 pg/mL) and remained elevated at finish (180 pg/mL: P < .01), with no significant differences between midpoint and race finish (Fig 1B). The median serum IL-6 (start 49.5 pg/mL, midpoint 40.8 pg/mL, and finish 42.0 pg/mL), IL-15 (start 56.9 pg/mL, midpoint 45 pg/mL, and finish 53.3 pg/mL), and IL-8 (start 3923 pg/mL, midpoint 3510 pg/mL, and finish 3764 pg/mL) concentrations did not differ significantly between the start, midpoint, and finish of the race (Fig 1C–E). Only 1 data point from IL-6 in a single dog was below the lower limit of detection. Measured TNF-α concentrations did not reach the detectable limit of the assay on at least 1 data point or more for 20 of the 28 dogs and a value of 0.16 pg/mL was given to these data points. There were no significant differences between median serum TNF-α concentrations across time points (Fig 1F).
Regression analysis was performed to examine the correlation between serum IL-6 concentrations and serum MCP and CRP concentrations at midpoint and finish. IL-6 and CRP showed no significant associations (midpoint, R = 0.08; finish, R = 0.13), whereas there were associations between IL-6 and MCP-1 at both the midpoint and finish (R = 0.66, P < .01; R = .88, P < .01: Fig 2A and B, respectively). Further examination of the association between MCP-1 and CRP at midpoint and finish showed no significant associations (R = 0.33, P = .10; R = 0.22, P = .28, respectively). Regression analysis between body weight and IL-15 showed no significant associations at midpoint (R = 0.03) and finish (R = 0.02).
Figure 2. A. Regression analysis of MCP-1 and IL-6 from serum samples obtained at midpoint. A significant positive correlation was observed (P < .01, r = 0.66). B. Regression analysis of MCP-1 and IL-6 from serum samples obtained at race finish. A significant positive correlation was observed (P < .01; r = 0.88).
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The sled dogs included in this study did not show significant systemic changes in serum myokine concentrations during or immediately after a 1,650-km race. These data are in accordance with a previous study, which showed no significant changes in serum IL-6 concentration in sled dogs after a 563-km race. The lack of systemic increased in IL-6 might reflect metabolic differences between endurance trained sled dogs and human endurance athletes. Exercising sled dogs are able to utilize primarily fat and protein as their fuel source and therefore glycogen depletion, which has been associated with increased serum IL-6, might not be as prevalent as in human endurance athletes.[5, 16]
No significant changes in serum concentrations of IL-15 and IL-8 were identified in the dogs enrolled in this study. These findings might be explained by the locally directed autocrine and paracrine activity of these myokines such that increased physical activity is not reflected by changes in systemic circulation.[5, 9] IL-15 increases have been associated with muscle hypertrophy while decreases are associated with atrophy. Although we expected a decrease because of the overall weight loss in the dogs, other factors might be involved in skeletal muscle IL-15 secretion being increased with increased muscle tissue fat oxidation. Therefore, in a species that preferentially utilizes fat more efficiently, IL-15 might not increase substantially in response to exercise. The data suggest that in sled dogs, these myokines have limited value as markers of metabolic changes influenced by endurance exercise. In addition, considering our timing for sample collection was within 4–6 hours of the cessation of exercise, it is entirely possible that there might have been modest increases or decreases in cytokines that were missed, and further investigation where blood is drawn at the cessation of an exercise bout might be far more revealing.
Our APR data are in agreement with a previous study demonstrating increased serum CRP concentrations in sled dogs after a 563-km and 1,850-km race.[2, 3] Distance trained dogs also appear to have increased baseline CRP values when compared to normal canine values and baseline values established in our laboratory for healthy house pets.[2, 3] The exact reasons for the higher baseline concentrations might be attributable to all teams having run within 48 hours of the prerace serum collection, which might have induced a mild acute phase response. Similar increases into the 10–25 μg/mL range have been observed in sprint sled dogs 24 hours after participating in a 16-mile sprint race. One would also expect that other inciting causes, such as infection and inflammation related changes in IL-6 or TNF-α, might induce this CRP response; however, the lack of a detectable response of TNF-α and lack of detectable changes in IL-6 indicates that infectious or serious exercise-induced inflammation is not the source of elevated CRP.[18, 19] Additional investigation is needed to determine the mechanisms of induction of the APR, which do not appear to be related to IL-6, as has been suggested in human endurance athletes.
Another component of acute low grade muscle inflammation is the induction of MCP-1, which was increased at both the midpoint and at the race finish, indicating that there is a mild sustained inflammatory response. Because of the lack of a TNF-α response, we conclude that the inflammatory response is most likely originating from the skeletal muscle, which is commonly observed in human athletes undergoing endurance exercise. Muscle damage is followed by a repair process involving the migration of macrophages into the muscle which can lead to a low mild increase in IL-6 attributable to release from macrophages. Interestingly, there was a significant positive correlation between IL-6 and MCP-1 at both the half-way point and race finish. Considering these elevations, the use of MCP-1 or possibly IL-6 as markers of muscle inflammation in sled dogs might be a fruitful area of investigation in dogs suspected of acute/subacute rhabdomyolytic events.
In conclusion, the serum myokines often associated with endurance exercise in human athletes do not appear to be elevated systemically in canine endurance athletes, which may be attributable to inherent differences in metabolism and skeletal muscle biochemistry. In this and other studies, IL-6 was not associated with the APR as in some human studies, which might be because of either time of sample acquisition during exercise or a lack of a response in dogs. However, the increase in serum MCP-1 and CRP suggest that mild inflammation and APR are present in endurance dogs. Therefore, in hindsight, to fully examine the inflammatory response of exercise, it would have been useful to examine interleukin-10 and interleukin-4, which are involved in dampening inflammation and promoting regeneration in skeletal muscle during exercise. These cytokines might be playing a significant role in the inflammatory response and further investigations in endurance dogs should include these cytokines in the analysis. A more complete analysis of this nature may hold the key to understanding the inflammatory response observed in our study by helping to differentiate normal physiologic response to exercise versus inflammation unrelated to exercise, possibly shedding light on the APR of exercise.