Post-Exercise Skeletal Muscle Glycogen Related to Plasma Cytokines and Muscle IL-6 Protein Content, but not Muscle Cytokine mRNA Expression

Objectives The purpose of this study was to correlate post-exercise muscle glycogen levels with changes in plasma cytokine, and muscle mRNA cytokine expression and protein content. Methods Twenty-four male runners (age 36.5 ± 1.8 years, VO2max 60.0 ± 1.5 mL⋅kg−1 ⋅ min−1) ran twice (separated by 4 weeks) on treadmills to exhaustion at 70% VO2max (average time and distance of 2.24 ± 0.09 h and 24.9 ± 1.1 km). Muscle biopsies from the vastus lateralis and blood samples were collected before and after each run, with IL-6, IL-8, and MCP-1 measured in muscle (mRNA and protein) and plasma. Data from the two runs were averaged. Results Participants experienced a 35.3 ± 4.2% decrease (P < 0.001) in skeletal muscle glycogen content (67.5 ± 2.8 to 44.3 ± 3.7 mmol⋅kg−1 wet weight). Muscle mRNA expression for IL-6, IL-8, and MCP-1 increased 7.34 ± 0.90-, 13.9 ± 2.3-, and 4.10 ± 0.60-fold, respectively (all, P < 0.001). Skeletal muscle IL-6, IL-8, and MCP-1 protein content increased 35.8 ± 10.6, 80.6 ± 12.1, and 105 ± 17.9%, respectively (all, P ≤ 0.005). Plasma IL-6, IL-8, and MCP-1 increased 47.1 ± 10.0-, 2.6 ± 0.3-, and 1.6 ± 0.1-fold, respectively (all, P < 0.001). Post-exercise muscle glycogen concentrations were negatively correlated with run time to exhaustion (r = −0.70, P < 0.001), and changes in muscle IL-6 protein content (r = −0.44, P = 0.049), plasma IL-6 (r = −0.72, P < 0.001), IL-8 (r = −0.60, P = 0.002), and MCP-1 (r = −0.589, P = 0.002), but not with changes in muscle IL-8 and MCP-1 protein content, or muscle mRNA expression for IL-6, IL-8, and MCP-1. Conclusion Prolonged and intensive running increased muscle mRNA expression, muscle protein content, and plasma levels for IL-6, IL-8, and MCP-1, and post-run muscle glycogen levels were most strongly related to plasma cytokine levels.


Introduction
Cytokines are a broad category of small proteins that are important in cell signaling, act through receptors, and affect the behavior of other cells. Cytokines are released by a range of immune cells, endothelial cells, adipocytes, muscle cells, and other cells, and include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor (1)(2)(3). Considerable attention has focused on the potential for regular exercise to counteract a range of disease states by stimulating cytokine production and release (2,3).
In response to published reports that carbohydrate ingestion during prolonged exercise attenuated the plasma IL-6 response, Steensberg et al. (26) hypothesized that a link may exist between low muscle glycogen content and IL-6 production during exercise. Using a two-legged knee-extensor exercise design, IL-6 mRNA levels and IL-6 release were shown to be augmented in the~40% glycogen-depleted leg compared with the control leg in seven men who exercised for 5 h. Keller et al. (27) showed that muscle IL-6 mRNA and transcriptional activity were enhanced when six male participants engaged in 3 h of two-legged knee-extensor exercise with low muscle glycogen levels. Chan et al. (13) reported that exercising (60 min cycling,~70% VO 2max ) in a glycogen-depleted state increased muscle IL-6 and IL-8 mRNA expression, with slightly higher plasma IL-6 but not IL-8 levels. Other studies have not been able to show that variance in muscle glycogen depletion with exercise is related to muscle IL-6 or IL-8 mRNA expression (11,14,15). Helge et al. (15) showed that IL-6 release is markedly higher from the exercising arms than the legs during wholebody exercise, and is not related to muscle glycogen utilization. Thus, the role of glycogen availability in the contracting muscle as a primary signaling mechanism for cytokine mRNA expression and release into the circulation is unclear, and interpretation has been made more difficult through research designs utilizing small subject numbers and modest exercise workloads (14)(15)(16)(17).
The purpose of this study was to relate post-exercise muscle glycogen levels with muscle mRNA IL-6, IL-8, and MCP-1 expression and protein content, and change in plasma cytokine levels in 24 male runners who ran twice on treadmills to exhaustion at~70% VO 2max . Muscle biopsies from the vastus lateralis and blood samples were collected before and after each run, and data from the two runs were averaged to strengthen data quality, reduce random measurement error, and provide better estimates of the "true" value for individual participants (28).

Participants
Subject recruitment was conducted via direct messaging to runners and running clubs in the Charlotte, NC, USA, metropolitan area. Participants included 24 male runners (ages 27-49 years) who regularly competed in long-distance road races and were capable of running at~70% on treadmills to exhaustion. During the study, participants consented to train normally, maintain weight, and avoid the use of large-dose vitamin and mineral supplements (above 100% Daily Value), and all herbal supplements and medications (in particular, non-steroidal anti-inflammatory drugs) for the 4-week period separating the two lab sessions. Participants signed informed consent forms and study procedures were approved by the Institutional Review Board at Appalachian State University (ASU).

Baseline Testing
Two weeks prior to the first running trial in the ASU Human Performance Laboratory, participants were tested for VO 2max during a graded, treadmill test with the Cosmed Fitmate metabolic system (Cosmed, Rome, Italy) (29). Body composition was measured with the Bod Pod body composition analyzer (Life Measurement, Concord, CA, USA). Demographic and training histories were acquired with questionnaires.

Running Trials
During the 3-day period prior to each running trial (separated by 4 weeks), participants followed a moderate-carbohydrate dietary regimen (~55% kcal as carbohydrates) by choosing foods from a list provided by the investigative team. A standardized meal consisting of Boost Plus (Néstle Nutrition, Slorham Park, NJ, USA) was ingested at 12:00 p.m., with energy intake adjusted to 5 kcal·kg −1 body weight. Boost Plus is a nutritionally complete, high-energy oral supplement with an energy density of 6.4 kJ·mL −1 (1.52 kcal·mL −1 ) and 15% of energy as protein, 35% as fat, and 50% as carbohydrate, and 24 vitamins and minerals. Participants reported to the lab at 2:15 p.m. and provided blood and muscle biopsy samples. At 3:00 p.m., participants ran on laboratory treadmills with the speed set at 70% of VO 2max , and drank water ad libitum, without ingestion of any other beverages or food. Participants ran as long as possible until exhaustion, defined as the inability of the subject to continue running at 70% VO 2max despite verbal urging from the laboratory staff. Metabolic measures from the Cosmed Fitmate metabolic system and the rating of perceived exertion (RPE) were taken at 15 min, and then every 60 min during the running bout to verify that the appropriate intensity was maintained. Blood and muscle samples were taken again immediately following exercise. These procedures were repeated 4 weeks later, with runners following the exact same schedule (including same time and day of the week), and running at the same treadmill speed to exhaustion.

Cytokine Analysis
Total plasma concentrations of three cytokines [monocyte chemoattractant protein-1 (MCP-1), IL-6, and IL-8] were determined using an electrochemiluminescence-based solid-phase sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD, USA). All samples and provided standards were analyzed in duplicate, and the intra-assay CV ranged from 1.7 to 7.5% and the inter-assay CV 2.4-9.6% for all cytokines measured. Pre-and postexercise samples for the cytokines were analyzed on the same assay plate to decrease inter-kit assay variability.

Stress Hormone Analysis
Serum cortisol was measured with an electrochemiluminescence immunoassay (ECLIA), and plasma epinephrine with highpressure liquid chromatography (HPLC) and electrochemical (EC) detection through a commercial lab (LabCorp, Burlington, NC, USA).

Muscle Biopsy Procedures and Glycogen Analysis
Pre-and post-exercise muscle biopsy samples were acquired from the vastus lateralis on the same leg~2 cm apart using procedures previously described (30). During the second running trial, the same procedures were followed, but on the opposite leg. Local anesthesia (1% xylocaine, Hospira, Inc., Lake Forest, IL, USA) was injected subcutaneously. After a small incision, a muscle biopsy sample was obtained using the suction-modified percutaneous needle biopsy procedure (30). Muscle was trimmed of connective tissue and fat and immediately frozen in liquid nitrogen. Samples were stored at −80°C until subsequent analysis. A glycogen assay kit (Catalog #MAK016, Sigma-Aldrich, St. Louis, MO, USA) was used to determine the concentration of glycogen in vastus lateralis muscle homogenates. In this coupled enzyme assay, glucoamylase hydrolyzed glycogen to glucose, and then the glucose was oxidized to yield a product that reacted with a probe to generate a color detectable with a microplate reader (Synergy H1 Hybrid Reader, BioTek Instruments, Inc., Winooski, VT, USA) at 570 nm.

Muscle mRNA Measurements
Total RNA extraction of vastus lateralis biopsies was conducted using an RNeasy ® Fibrous Tissue Mini Kit (Qiagen, Limburg, Netherlands) according to the manufacturer's instructions. Frozen muscle biopsies were homogenized in RTL-βmercaptoethanol Buffer with a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). An aliquot of RNA from all samples were run on 1.5 denaturing agarose gels stained with ethidium bromide to confirm RNA integrity. Additionally, RNA quality was further verified spectrophotometrically using the A 260 /A 280 ratio which was determined using a nanodrop. Reverse transcription of RNA into cDNA was conducted using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) as per manufacturer's instructions. Reverse transcription was per-  (31). Changes in cytokine expression post-exercise were normalized to pre-exercise levels.

Statistical Analysis
Data are expressed as mean ± SE. Dependent t-tests were used to test change (pre-to post-exercise), with the level of significance set at P < 0.01. The Pearson product-moment correlation coefficient was used as a measure of the linear correlation between two variables.

Results
Participants included 24 trained male runners (ages 22-55 years) who successfully adhered to all aspects of the study design (see subject characteristics in Table 1). Run distances to exhaustion and post-exercise muscle glycogen content for the two run trials were significantly correlated (r = 0.74, P < 0.001; r = 0.67, P < 0.001, respectively), as were between trial post-run plasma cytokines levels (r = 0.475, 0.545, and 0.584 for plasma IL-6, IL-8, and MCP-1, respectively, all, P < 0.05). Between trial correlations for muscle cytokine mRNA and protein levels were non-significant. Metabolic and performance data from the average of the two run-to-exhaustion trials are summarized in Table 2. The runners were able to maintain an average intensity of 69.2 ± 1.3% VO 2max for 2.24 ± 0.09 h and 24.8 ± 1.1 km. The RPE was 17.8 ± 0.2 at the end of the running trials.

Discussion
As expected, these data from 24 male endurance athletes who ran twice on treadmills to exhaustion at~70% VO 2max showed increases in muscle IL-6, IL-8, and MCP-1 mRNA and protein content, and corresponding increases in plasma levels for these three cytokines. This study focused on the linkage between muscle glycogen availability and cytokine mRNA expression and production, and showed that post-run muscle glycogen content was negatively related to plasma but not muscle mRNA levels for IL-6, IL-8, and MCP-1. No significant correlations were found between exercise-induced changes in stress hormones and plasma or muscle cytokines (both mRNA and protein levels), similar to results previously reported by our research group (10,11). The correlation between low muscle glycogen content and enhanced muscle cytokine mRNA expression is not a consistent finding, and depends on the study design, with the strongest support demonstrated when participants initiate exercise with low muscle glycogen levels (13,26,27). When participants engage in prolonged, intensive exercise with normal pre-exercise glycogen levels, depletion in muscle glycogen stores varies widely between participants and typically cannot be linked to muscle IL-6 or IL-8 mRNA expression (10,11,(14)(15)(16). Moreover, data from our laboratory indicate that even though cycling induces greater vastus lateralis muscle glycogen depletion in comparison to running, muscle cytokine mRNA expression is multiple times greater with running (10,11). IL-6 release is markedly higher from the arm compared with the leg during whole-body exercise, and is not linked to muscle glycogen content (15). Additionally, carbohydrate compared to placebo ingestion during prolonged and intensive exercise increases blood glucose levels and decreases muscle mRNA expression and plasma levels for IL-6 and IL-8 despite similar muscle glycogen depletion rates (10,11). In the current study, subjects drank water ad libitum during the mid-afternoon running trials (3-h post-prandial state), without ingestion of any other beverages or food. Thus carbohydrate ingestion would have been expected to attenuate post-exercise increases in plasma IL-6 and IL-8, perhaps weakening the linkage to decreases in muscle glycogen content. Although the data are not entirely consistent, regular exercise bouts in the fasted state without oral carbohydrate intake may enhance training adaptations (32,33). This is the first study to simultaneously measure muscle mRNA expression, muscle protein content, and plasma levels for three cytokines (IL-6, IL-8, and MCP-1) after exhaustive exercise.
Interpretation of the data from this study was strengthened by combining results from two intense, long duration exercise sessions for 24 male runners (28), in contrast to other studies utilizing small subject numbers and modest exercise workloads (14)(15)(16)(17). While others have shown increases in skeletal muscle MCP-1 mRNA following exercise (8), this study demonstrated that postexercise increases in both muscle MCP-1 mRNA expression and protein content are unrelated to post-exercise muscle glycogen levels. Della Gatta et al. (8) showed that muscle mRNA expression and protein content for IL-6, IL-8, and MCP-1 were increased in biopsy samples obtained from eight untrained men 2 h following intensive leg resistance exercise. Plasma cytokine levels were not reported in this study, but data suggested that MCP-1 and IL-8 were expressed by cells in the interstitial space between muscle fibers, including macrophages, satellite cells, and other stromal cells (8). A limitation in the current study is that repeated muscle and blood samples were not collected during the first several hours post-exercise that may have revealed additional insights.
Taken together, under the normal context within which athletes train and compete, muscle glycogen depletion at best represents just one potential signal for cytokine mRNA expression within muscle tissue. Other signaling mechanisms may include free fatty acid availability, blood glucose levels, nitric oxide, and exerciseinduced muscle damage and adaptation (8,11,16,20). Nonetheless, our data support a significant, modest, negative relationship of post-exercise muscle glycogen and plasma cytokine levels (13), and this could be due in part to the release of IL-6, IL-8, and MCP-1 from extra-muscular sources, including adipose tissue (3,16) and the brain (7). Additionally, our data support a significant, negative relationship between post-exercise muscle glycogen content and both muscle and plasma IL-6 protein levels, but not muscle IL-6 mRNA expression, suggesting that unmeasured factors within the secretome of the working skeletal muscle may differentially influence cytokine mRNA stability, transcription, production, degradation, modification, and release into the circulation (12,13).

Funding
Reoxcyn Discoveries Group, Salt Lake City, UT, USA.