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C. Demczar1, C. Behrens2, L. Baltz2, V.P. Georgescu1, D. Arminavage1, J.M. Brown1, D. Fussell1, T.C. Trate3, S.R. McAnulty1, L.S. McAnulty2, E.K. Merritt1


1. Department of Health and Exercise Science, Beaver College of Health Sciences, Appalachian State University; 2. Department of Nutrition and Health Care Management, Beaver College of Health Sciences, Appalachian State University; 3. Appalachian Rehabilitation, Boone, NC

Corresponding Author: Edward K. Merritt, ASU Box 32071, 111 Rivers St., Boone, NC 28608, Phone: (828) 262-7986, Fax: (828) 262-3138, merritte@southwestern.edu

J Aging Res Clin Practice 2017;6:229-237
Published online November 23, 2017, http://dx.doi.org/10.14283/jarcp.2017.31



Muscles of old adults respond to stress with heightened inflammatory signaling that disrupts the regenerative process. This muscle inflammation susceptibility could contribute to the age-related decline in muscle mass, as anti-inflammatory medications taken concurrently with exercise training, have proven beneficial in attenuating age-related loss of muscle mass. With antioxidants and anti-inflammatory potential, blueberries (BB) are a natural alternative that might regulate aged muscle inflammation susceptibility. Objectives: The purpose of this study was to determine the effects of BB consumption on the muscle inflammatory profile of older adults, and to determine the subsequent muscle inflammatory response to exercise. We hypothesized that BB would lower the inflammatory profile of muscle and attenuate the inflammatory response after resistance exercise. Design: Subjects were randomized to receive daily BB or placebo supplements, which were blind to subjects and researchers. All subjects underwent baseline functional testing, post-supplementation testing, and testing post-muscle stress stimulus. Setting: Volunteers were recruited from Western North Carolina region, USA. Participants: Healthy, non-resistance trained adults over 60 years old (n=22) were recruited. Measurements: Profiles of inflammation pathways known to affect muscle mass were established prior to and after 6-weeks of daily consumption of BB. Post-supplementation, subjects performed an exercise protocol to induce inflammation and returned 24 hours post-exercise to determine the muscle inflammatory profiles. Results: Muscle cytokine and soluble cytokine receptor levels were similar between groups and within groups before and after BB consumption. Cytokine and cytokine receptor levels post-muscle stress changed similarly in the BB and placebo group, indicating BB had no effect on the muscle’s inflammatory response. Total plasma antioxidant capacity was 22% higher in the BB group 24-hours post-muscle stress, however, plasma oxidative stress was not different between groups or within groups. Conclusion: While BB consumption did not affect inflammatory signaling pathways within the muscle nor affect inflammation after a regenerative stimulus, a higher plasma antioxidant capacity could contribute to a better long-term regenerative response.

Key words: Skeletal muscle, inflammation susceptibility, blueberries, aging, anti-inflammatory.




The age-related loss of skeletal muscle mass, termed sarcopenia, occurs gradually over time and is strongly associated with a progressive decline in physical capacity and can lead to disability and loss of functional independence. The etiology of aging atrophy is poorly understood. Among the potential causes that continue to be explored, an area of interest involves muscle regenerative biology. Aging atrophy might in part result from episodes of incomplete muscle repair throughout adulthood. Whether this is a major cause of atrophy of the normal aging muscle is debatable, but there is no question that skeletal muscle regenerative capacity declines with age as is consistently shown following overt muscle damage (1, 2).
Based on research findings to date, it is theorized that muscles of old (vs. young) suffer heightened and prolonged pro-inflammatory and proteolytic signaling that disrupts the local environment leading to failed myogenesis, and ultimately, a decline in muscle mass for a given concentration of cytokines in the circulation or local interstitial compartment. Evidence has been obtained in vitro and in vivo, to further suggest that this is the case. Skeletal muscle from healthy, older humans has an elevated local inflammatory profile when compared to young, despite the lack of evidence of systemic inflammation (3, 4). Bolstering this theory is the evidence that pharmacological inhibitors of the cyclooxygenase inflammatory pathway enhance gains in muscle size and strength in older individuals undergoing resistance training, but not young adults (5). Additionally, in vitro, muscle progenitor cells from older adults respond with a heightened inflammation response compared to younger adults when subjected to an identical concentration of inflammatory cytokines. These findings have broad implications for aged skeletal muscle research and might help explain the cause of sarcopenia as well as the blunted regenerative response to injury that has been documented in older adults (4).
Preventing local muscle inflammation is an obvious way to further test the aged muscle inflammation susceptibility theory. Blueberries, with high concentrations of antioxidants and anti-inflammatory compounds such as anthocyanins, are an ideal natural, non-pharmacologic candidate to test the theory. Evidence exists that other fruits with anti-inflammatory compounds are beneficial for muscle recovery following damage in young adults [6]. However, it is unknown how the muscle’s inflammatory profile and cellular response to damage were affected, nor is it known if the fruit would benefit older adults in a similar manner. Therefore, the purpose of this study was two-fold: 1) To determine the effects of 6 weeks of blueberry supplementation on the systemic and local muscle inflammatory profile of older adults; and 2) To measure the inflammatory/regenerative response of the muscle following a regenerative stimulus with or without blueberry supplementation. We hypothesized that 6-weeks of blueberry supplementation would lower the basal inflammatory profile of aged skeletal muscle, and would subsequently attenuate the heightened inflammatory response normally observed after a stressful bout of resistance exercise.


Materials and Methods


Twenty-four men and women over 60 years old were recruited to partake in this study (Table 1). Volunteers were screened with a health history questionnaire and allowed to participate if they were apparently healthy and did not participate in a regular exercise program that included any form of resistance exercise or high impact aerobic activity such as running for six months prior to the study and for the duration of the study  Subjects were excluded from the study if they had a body mass index greater than 30, used any type of anti-inflammatory medications or suffered from a disease characterized by a chronic inflammatory state, had uncontrolled, severe hypertension, a lidocaine allergy, or were told by their doctor that they should not engage in strenuous exercise. The subjects further agreed not to alter any lifestyle habits during the study, as well as refrain from daily use of non-steroidal anti-inflammatory drugs or other medications that may impact any aspect of the study. All subjects signed a written informed consent document approved by the local Institutional Review Board, and were informed of all protocols, procedures, and potential risks associated with the study.


Table 1 Subject descriptives Pre/Post-supplementation

Table 1
Subject descriptives Pre/Post-supplementation


Study Design

This study consisted of three visits, the second occurred six weeks after the first visit, and the third occurred 24 hours following the second visit (See Figure 1). During visit 1, informed consent and the medical screen were completed, followed by baseline measurements. These included height, weight, body composition, a blood draw, muscle biopsy, and strength measurements (described below). In addition, diet recording instructions were explained to the subject, where the first diet baseline was taken three days prior to starting supplementation. Subjects were randomized to the blueberry or placebo group. Between visits 1 and 2, the subjects consumed the supplement or placebo. Following the 6 week supplementation period, the subjects returned for a visit 2. During visit 2, anthropometric measures and diet records from the previous week were collected. A blood draw and muscle biopsy were then performed. The subject then underwent a mechanically-induced muscle stress protocol as a stimulus for inflammation and subsequent regeneration. Subjects returned to the laboratory for visit 3, 24 hours after visit 2. The final blood and muscle biopsy samples were obtained at this time.

Body Composition Assessment

Dual energy X-ray absorptiometry (DXA: Discovery W (S/N 81225); Hologic, Inc. Marlborough, MA) was performed to determine whole body fat and lean mass, thigh muscle mass, and body fat percentage according to manufacturer’s instructions.

Figure 1 Study timeline for subjects from informed consent to last visit

Figure 1
Study timeline for subjects from informed consent to last visit


Strength Measurements

Two strength measurements were taken during visit 1. A maximal isometric knee extension utilizing the right leg of each subject was taken using a transistor that was amplified by and converted by a DAQ board (National Instruments, Austin, TX) to give a quantitative output in Newtons (LabView, National Instruments, TX). Up to five trials were recorded, with the maximum value obtained being considered as the subjects isometric knee extension maximum force. The second strength measurement was a one repetition maximal effort knee extension exercise completed on a Cybex Isotonic Knee Extension machine. Initial resistance was estimated using the isometric strength value converted to pounds, and weight was added until a single repetition could not be completed with proper form. This value was considered the subject’s knee extension one repetition maximum and was used to determine the resistance used for the muscle stress protocol (60% of maximum).

Diet Education/Analysis

Each subject was informed on how and when to accurately record dietary intake on a three-day diet record during the first and final week of supplementation.  On visit 2, the returned diet records were reviewed with the subject to ensure completeness and clarity.  Data from diet records were entered into diet analysis software (The Food Processor SQL – Version 10.12.0, ESHA Research, Salem, Oregon).  Detailed printouts of nutrient intake were compared to written entries by another member of the research team to identify errors in entry and ensure accurate data entry.  Data from three-day diet records were analyzed to determine differences in dietary intake within and between groups at baseline and 6-weeks post-supplementation.

Blueberry/Placebo Supplementation

The supplementation protocol consisted of subjects ingesting 100% freeze-dried blueberry powder [U.S. High Bush Blueberry Council (USHBC), Folsom, CA, 50/50 blend of Tifblue (Vaccinium virgatum [ashei]) and Rubel (Vaccinium corymbosum)] or placebo (maltodextrin, fructose, artificial flavoring, artificial purple and red color, citric acid, and silica dioxide) daily for six weeks. Subjects consumed 38 g of powder, equivalent to approximately 250 g of whole blueberries. Specific nutritional data for the powders was previously reported (7).  Blueberry and placebo packets coded by the USHBC to blind researchers and subjects to their assignment were apportioned into labeled week-by-week bags. Subjects were instructed to consume packets with 8-ounces of water with their evening meal, but were told to avoid consuming dairy products with the supplement. Compliance was confirmed via weekly e-mail/phone correspondence, and subjects were instructed to return supplement packaging, empty or otherwise, to its respective weekly bag for return at the conclusion of the study. Upon return, packets were counted to determine compliance.

Blood and Muscle Sampling

Blood sampling occurred in the morning when the subjects were in a fasted, rested state. Approximately 10-ml of blood was collected from an antecubital vein into heparinized and EDTA vacutainer tubes. The tubes were immediately placed on ice and then spun at 1000g for 10 min at 4 °C. The plasma from the heparin tubes was aliquoted into cryotubes, frozen in liquid nitrogen, and stored at –80 °C until analyses.
Muscle biopsies were taken immediately following the blood draws. A total of three biopsies were obtained, one during each visit; visit 1, before supplementation, visit 2, after the 6-weeks of supplementation, and visit 3, 24-hours post muscle stress exercise. Muscle samples were taken from the m. vastus lateralis under local anesthetic (1% lidocaine) by percutaneous needle biopsy. The contralateral limb was used for the post-muscle damage biopsy.  Samples were snap frozen in liquid nitrogen and stored at -80°C until analysis.

Mechanically-Induced Muscle Damage

Subjects performed 9 sets of ten repetitions of a bilateral knee extension exercise using a resistance equivalent to 60% of that subject’s one repetition maximum (1RM), as determined by the isotonic knee extension strength measurement. Similar protocols were found sufficient to induce an inflammatory response and moderate damage to myofibers in untrained subjects (4). Subjects were instructed to perform the isotonic knee extension emphasizing a fast concentric phase and a slow eccentric phase at approximately a 1:4 time ratio. A one minute rest period was given between sets. If the subject was unable to finish all ten repetitions of the previous set a longer rest was allowed rather than lowering the weight.

Plasma oxidative stress measurement

F2-isoprostanes were determined using an enzyme linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions (Cayman Chemical #516360, Ann Arbor, MI).  Briefly, this is a competitive assay with a range of 2.5 to 1,500 pg/mL and a sensitivity (80% B/Bo) of about 10 pg/mL.

Plasma antioxidant potential

Total plasma antioxidant potential was determined by the ferric reducing ability of plasma (FRAP) assay according to the methodology of Benzie et al. (8). The basis of this assay is that water soluble reducing agents (antioxidants) in the plasma will reduce ferric ions to ferrous ions, which then react with an added chromogen. Samples and standards were analyzed in duplicate and expressed as ascorbate equivalents based on an ascorbate standard curve (0-1000 µmol/L). Intra-assay and inter-assay coefficients of variation were less than 5% and 7%, respectively.

Plasma creatine kinase activity

Plasma creatine kinase activity was measured on visit 2 and visit 3 samples using a commercially available kit following the manufacturer’s instructions (Sigma-Aldrich, # MAK116, St. Louis, MO).

Muscle Protein Isolation

Snap-frozen muscle samples (~30 mg) were homogenized following a 15 minute pre-incubation in 6 ul/mg muscle of ice cold lysis buffer with phosphatase inhibitors (Milliplex, Billerica, MA, USA; #43-040) with AEBSF protease inhibitor added (Milliplex; #101500) and then centrifuged at 14,000xg for 2 x 20 min at 4°C and assayed for protein content using the bicinchoninic acid (BCA) technique with BSA as a standard (Bio-Rad, Hercules, CA). Supernatant was stored at -80°C until further analysis.

Muscle Inflammatory/Cell Signaling Analysis

Twenty-five ug of total protein was resolved on 4-12% NuPAGE Bis-Tris gels (Novex, Life Technologies) and transferred overnight onto PVDF membranes (Bio-Rad, Hercules, CA).  Immunoprobing was completed with antibodies from Cell Signaling Technologies (Danvers, MA).  Antibodies against proteins known to be involved in the pro-inflammatory signaling cascade in muscle were used, including, TWEAK, TWEAK Receptor, Fn14, SOCS3, p50/p105 NFκβ, STAT3, phospho-STAT3 (Ser727), HSP27, and HSP70. HRP-conjugated secondary antibody (Pierce Thermo Scientific, Rockford, IL) was used at 1:2000 (w/v) followed by chemiluminescent detection. Bands were detected by chemiluminescence in a Bio-Rad (Hercules, CA) ChemiDoc XRS+ imaging system, and densitometry was performed using Bio-Rad analysis software.

Cytokine Protein Analysis


Twenty ug of each homogenized muscle sample were loaded in duplicate onto plates for multianalyte profiling of cytokines using the Magpix (Luminex, Austin, TX) multiplex platform. Cytokines and soluble cytokine receptors (GCSF, IFNα2, IL10, IL13, IL15, IL1a, IL4, IL5, IL6, IL7, MCP1, GP130, IL6 Receptor, TNF Receptor 1, TNF Receptor 2) in muscle homogenates were measured using the MILLIPLEX® MAP assay kit (Millipore, #HCYTOMAG-60K) according to manufacturer’s specifications and analyzed using Milliplex Analyst software.


Twenty-five ul of plasma from each sample were loaded in duplicate onto plates as above to determine multianylate profiling of specific cytokines (IL8, IL10, MCP1, TNFα).

Statistical Analysis

All data are expressed as means ± SEM unless otherwise noted. Student’s T-tests were used to analyze between group differences in subject descriptives. Outcome measures were analyzed using a 2 (groups) × 3 (times) repeated measures ANOVA. Main effects of treatment, time, and treatment–time interaction were determined by the method of Greenhouse–Geiser. If significant treatment by time interaction was detected, differences between and within treatments for specific times were analyzed with pairwise comparisons with significance set at p ≤ 0.05 after Bonferroni correction to account for multiple comparisons. Cohen’s effect size (d) was calculated to determine the magnitude of changes over time or between conditions and assessed as 0.2 = small effect, 0.5 = moderate effect, and 0.8 = large effect. Only changes with large effect sizes (where main effects were P > 0.05 < and P ≤ 0.10) are included in the results and discussion.



Subjects included 24 volunteers (ages 60 – 79 years) who were enrolled in the study. One subject withdrew prior to visit 1 and a second subject withdrew immediately prior to visit 2, both for non-study related reasons. Data from these subjects has been excluded from analyses. The remaining 22 subjects completed the entire study (Table 1). No significant differences existed between groups or between pre- and post-supplementation physical characteristics.
Compliance, as measured by packet return (both empty and full), was over 97% with no single subject returning less than 94% of the packets. Diet records indicated no significant differences within groups over time or between groups for macronutrient composition (Supplementary Data Appendix A).
Dietary selenium pre-supplementation (60.3 + 38.5 ug/day) vs. post-supplementation (48.9 + 30.7 ug/day) within the blueberry group and dietary copper pre (1.0 + 1.2 mg/day) vs. post (0.7 + 0.4 mg/day) within the placebo group declined significantly (p < 0.05). No within or between group differences existed for any other micronutrients.
Cytokine and soluble cytokine receptor levels measured from the muscle biopsy sample were not significantly different between groups or within groups between visit 1 and visit 2, with one exception (Supplementary Data Appendix B). Muscle IL-10 levels were significantly higher at visit 1 in the placebo group compared to the blueberry group (p <0.05), but no differences existed post-supplementation or post-muscle stress at visits 2 or 3 between or within either group.
Plasma creatine kinase activity levels, an indicator of skeletal muscle injury, were 34.4 ± 8.3 units/L in both groups combined at visit 2 and significantly increased to 60.8 ± 10.0 units/L at visit 3, 24-hours post-muscle stress, an increase of 77%, however the magnitude of increase was the same in both groups. Similarly, from the muscle biopsy samples, monocyte chemotactic protein-1 (MCP-1) and TNFα Receptor 1 significantly increased from visit 2 (pre-muscle stress) to visit 3 (24 hr post-muscle stress) when groups were combined (Figures 2 & 3), but no between group differences existed. Soluble cytokine receptors including IL-6 receptor and TNFα receptors 1 & 2 were significantly higher at visit 3 in the placebo group than at visit 2, but no between group differences existed (Figures 2 & 3).

Figure 2 Muscle MCP1 levels at each visit for blueberry (BB) and placebo groups.

Figure 2
Muscle MCP1 levels at each visit for blueberry (BB) and placebo groups.

* denotes significant difference between timepoints 2 & 3 with groups combined. + denotes significant difference between timepoints 2 & 3 for respective group. P < 0.05.

Figure 3 Muscle soluble cytokine receptor levels for blueberry (BB) and placebo groups at each timepoint.

Figure 3
Muscle soluble cytokine receptor levels for blueberry (BB) and placebo groups at each timepoint.

* denotes significant difference between timepoints 2 & 3 with groups combined. + denotes significant difference between timepoints 2 & 3 for one group. P < 0.05.


As with the cytokines and receptors measured in the muscle, plasma cytokine and soluble cytokine receptor levels showed few differences due to blueberry supplementation (Supplementary Data Appendix C). Plasma IL-10 levels were significantly higher in the placebo group pre-supplementation at visit 1 (p < 0.05), but as in the muscle, no differences existed between groups at visit 2 or 3. Unlike the changes noted in the muscle due to the muscle stress stimulus in MCP-1 or the soluble cytokine receptors, no differences existed in plasma cytokines and soluble cytokines from visit 2 to visit 3 nor between groups at either visit.
Of note, muscle levels of IL-6 were not significantly different between groups or within groups at any timepoint (Figure 4). Plasma levels of IL-6 were below detectable limits in most subjects at all timepoints. Muscle levels of TNFα were not detectable in any subjects at any timepoint, but plasma levels of TNFα were not significantly different between groups or within groups at any timepoint (Figure 5).

Figure 4 Muscle IL-6 levels for blueberry (BB) and placebo groups at each timepoint.

Figure 4
Muscle IL-6 levels for blueberry (BB) and placebo groups at each timepoint.

No significant differences between groups nor within subjects over time. P < 0.05. Note: Plasma IL-6 levels were undetectable


Western Blot analysis and multiplex cell signaling analysis of molecules involved in the IL-6 and TNFα skeletal muscle inflammation signaling cascade (SOCS3, NFkB, TWEAK, TWEAKR,) did not reveal any significant differences between groups nor within groups over time (Supplementary Data Appendix D). Additionally, no differences existed between or within groups in heat shock proteins 27, 60, 72, or 90a (Data not shown).
Total plasma antioxidant capacity as measured by FRAP was higher in the blueberry group than in the placebo group at visit 3 (Pearson’s Correlation p = 0.08, Cohen’s d = 0.85) (Figure 6). Plasma oxidative stress as measured by F2-isoprostanes was not different between groups or over time within groups.


Figure 5 Plasma TNFα for blueberry (BB) and placebo groups at each timepoint. Note: Muscle levels of TNFα were not detectable.

Figure 5
Plasma TNFα for blueberry (BB) and placebo groups at each timepoint. Note: Muscle levels of TNFα were not detectable.

No significant differences between groups nor within subjects over time. P < 0.05


Figure 6 Plasma FRAP for blueberry (BB) and placebo groups at each timepoint.

Figure 6
Plasma FRAP for blueberry (BB) and placebo groups at each timepoint.

‡ Denotes strong between group effect d > 0.8 & p < 0.10



The findings that basal, non-stressed muscles have a similar inflammatory profile whether or not blueberries have been consumed daily for the previous six weeks and that the inflammatory response to a muscle stress stimulus is similar between blueberry and placebo groups disproves the original hypothesis. Some cytokine and signaling proteins were significantly different between blueberry and placebo groups prior to supplementation at visit 1, but most were no longer significantly different between groups after supplementation at visits 2 or 3. If differences between groups still existed, supplementation had no significant effect within subjects when compared between groups. While these results were unexpected because it was theorized that true group differences due to supplementation would be identified, six weeks of blueberry supplementation does not appear to have any clinically significant effect on the skeletal muscle inflammatory profile of older adults, nor on their regenerative response to exercise-induced injury as measured at the 24-hour time point post-injury.
Despite the lack of differences between blueberry and placebo groups in plasma cytokines, muscle cytokines and inflammation signaling proteins, a strong effect (Cohen’s d > 0.8 where main effects were between p > 0.05 and p < 0.10) was noted in total plasma antioxidant capacity as measured by FRAP. Total plasma antioxidant capacity was higher in the blueberry group post-muscle stress than in the placebo group. Muscle damaging exercise, such as eccentric exercise, is known to increase oxidant stress [9], and the increased antioxidant capacity noted in the current study is similar to what has been observed after blueberry consumption in runners (10) and young females after eccentric muscle stress (11). However, this is the first time this has been documented in older adults following muscle stress. The ultimate effects of blueberry consumption on inflammation levels and muscle recovery is difficult to determine though, as studies have determined no additional benefit of supplementing with different antioxidants such as Vitamin C or E (12), but others have shown beneficial effects on muscle but not against the inflammatory response (13). Further studies have proven that blunting the oxidant and/or inflammation response to exercise by consuming antioxidants might actually be detrimental and prevent the positive adaptations induced by exercise (14), so it is not clear what impact an increased systemic antioxidant capacity would have on regeneration in this aged population.
Unfortunately, most studies researching consumption of anti-inflammatories or antioxidants prior to exercise have not considered the impact of aging on these processes. That older adults have a blunted muscle regenerative response is well-established. The reasons this occurs are less clear, but, in vitro observations of muscle satellite cells from elderly subjects have proven that they have a decreased antioxidant capacity (15). Satellite cells are of incredible importance in determining the success of the muscle’s regenerative response, so improving the antioxidant capacity in this case, could be beneficial. Further insight can be gained by studies such as that of Trappe et al. in which daily supplementation with ibuprofen and acetaminophen were beneficial to older adults in a resistance training program and allowed for greater increases in muscle strength and size (5). Oxidative stress can certainly contribute to the type of inflammation that ibuprofen is attenuating, so it stands to reason that if consuming blueberries is increasing antioxidant capacity in the elderly, a beneficial effect to the exercised/damaged muscle could exist and muscle regenerative capacity could be improved.
Several factors might be contributing to our inability to discern some of the hypothesized effects of long-term blueberry consumption on muscle inflammation susceptibility in older adults:

1) Subjects might have been too healthy to experience much benefit from an anti-inflammatory food being added to an already healthy diet in an already healthy, and possibly non-inflamed body.
Utilizing prescription of hypertension medications as a surrogate for general health, 26% of subjects in this study were on prescription hypertension medications, much lower than the average of 53% – 63% of older Americans within the same BMI range as our subjects (16). Also of note in relation to overall health, subjects in the current study were well educated with 70% having completed college (67% of placebo vs 73% of blueberry) compared to approximately 30-40% of Americans in this age group (17). Since fruit and vegetable consumption generally increases with education level (18), it is likely, as 3-day diet records showed, that subjects in this study generally eat more fruits and vegetables than the average person, and their baseline anti-inflammatory food consumption might already be enough to prevent the traditional aging muscle inflammation susceptibility noted in previous research (3). An additional change to subject exclusion criteria to exclude potential subjects who consume any nutritional supplements, or at least provide a “wash-out” period prior to enrollment in the study might have affected the results. For the current study, only potential subjects who were taking medications or supplements known to affect inflammation were eliminated, otherwise, subjects were told not to modify their intake of diet or supplements for the duration of the study. Nutritional supplements have a wide variety of purported effects, many of which are untested, but the possibility exists that some were already on an anti-inflammatory nutritional regimen and as such, saw no benefit to further increasing anti-inflammatory capacity.
The possibility exists that subjects who are already experiencing inflammation at clinically high levels would be the most likely to benefit from an anti-inflammatory treatment. The subjects in the current study were very healthy 60+ year old men and women recruited similarly to a previous study in which systemic inflammation was not detectable, but muscle inflammation was higher than in younger adults (3). Over 75% of potential subjects were not enrolled due to numerous health factors that made them ineligible for the current study, although many were eliminated due to high body mass index (BMI). Since normal aging is associated with increased systemic inflammation (19), and our subjects likely did not have increased systemic inflammation compared to young adults, perhaps less stringent exclusion criteria to include those who have a higher BMI would have affected the results. Including subjects with higher BMI would likely have lead to inclusion of subjects with higher fat mass, which contributes to inflammation (20). Higher levels of inflammation, systemically and possibly in the muscle, could prove necessary for the clinical significance of blueberries anti-inflammatory properties to be realized.

2) The timing (24-hr post-stress stimulus) was not ideal to measure the true inflammatory insult.
Due to the need to limit the number of muscle biopsies each subject received, the only measurement post-muscle stress was 24 hours after the eccentric exercise bout. This timepoint was chosen for several reasons including the need to be able to compare results to previous studies, such as the original aged muscle inflammation susceptibility theory paper (3) which observed changes in inflammatory signaling pathways 24-hours post-muscle damage. Other markers of muscle damage are increased immediately post-exercise but also upwards of 48 hours post-exercise, such as MCP-1 levels (21, 22). Muscle soreness and plasma creatine kinase, both crude measures of muscle damage and inflammation, generally peak 24-48 hours post-damage (23). Cytokines on the other hand, are more difficult to determine with some, such as IL-6, often peaking after only a few hours, and sometimes returning back to normal within 24 hours or sometimes remaining elevated for upwards of 72 hours depending on the subjects and protocol (24-26). Due to these factors, for a single post-muscle stress biopsy, the 24-hour timepoint was deemed a timepoint likely to capture the most differences in both inflammation signaling within the cells as well as cytokine levels in plasma and muscle samples. However, given the obtained results, perhaps earlier and/or a later timepoints would have elucidated more of the changes.
3) Anti-inflammatory effects only work acutely, and since the subjects had been 24 hours without consuming blueberries when they experienced the muscle stress stimulus, the effects of blueberries on skeletal muscle had already expired.
Research on the consumption of another fruit product (cherry concentrate juice or powder) has shown a positive impact on muscle health in younger, well-trained subjects. Cherry concentrate consumption increased the functional recovery after eccentric exercise induced muscle damage and similar results were seen in a later study with a powdered form (6, 27). The supplements were given in the days leading up to the muscle damage and the day of and the days after the muscle damage occurred. This is an important difference to note from the current blueberry study, as subjects in the current study had not consumed blueberries in the 24 hours prior to the muscle damaging stimulus and did not consume any afterwards.
Measuring functional recovery was not the goal of the current study, but if in fact the beneficial effects of cherries on muscle recovery post-damage are due to anti-inflammatory properties of the cherries as was implied, it might be necessary that supplementation occur acutely, immediately before and after the injury. If this is the case, it is likely that blueberries would have a similar if not more potent effect. Blueberries have more of the anti-inflammatory anthocyanins than cherries (28), but the cherry juice concentrate was reported to have a higher amount of anthocyanins than raw cherries. However, this amount is still lower than the amount of anthocyanins consumed by subjects in the current study (547 mg/day for cherry juice vs. 3597 mg/day for blueberries).

4) The muscle stress stimulus was overwhelming, and perhaps not even a potent pharmacologic anti-inflammatory agent could prevent the response.
Finally, the possibility exists that the muscle stress stimulus caused by high volume eccentric contractions was overwhelming and the anti-inflammatory effects of blueberry supplementation over the previous 6-weeks was not enough to counteract such a strong inflammatory insult. As discussed, the single timepoint post-stress muscle biopsy makes this difficult to determine, but evidence exists that the inflammatory insult was present 24-hours after muscle stress. MCP-1 and TNFR1 levels were significantly higher post-muscle stress (visit 3) compared to pre-muscle stress (visit 2) when both groups were combined, and IL6R and TNFR2 levels were significantly higher in the placebo group post-muscle stress. While this was not the case in the blueberry group, receptor levels in the blueberry group were not statistically different from those in the placebo group post-muscle stress. These results indicate that the muscle is responding to the insult, but perhaps it was too much of an insult for blueberry supplementation to significantly counteract.
An interesting way to change the stimulus to determine if blueberry supplementation has any true effects on muscle health in older adults would be to use low “dose” stress stimuli as are typically seen in a normal resistance training program. Whereas the current study utilized 9 sets of 10 repetitions with a focus on the eccentric (muscle lengthening), damaging phase, a standard resistance training protocol has 2-3 sets of 10 repetitions focusing on a balanced concentric/eccentric movement. As was proven by Trappe et al., older adults consuming a pharmacologic anti-inflammatory agent such as ibuprofen over 12 weeks while partaking in a long-term resistance training program is beneficial for muscle gains in strength and hypertrophy (5). In the study, subjects were undergoing a relatively lower muscle stress three times per week in the form of resistance exercise training. This stimulus is generally accepted to cause an inflammatory response (29), but the observation that the ibuprofen administration improved muscle hypertrophy and strength, implies that the ibuprofen was able to prevent too large of an inflammation response. Muscle regeneration between exercise sessions might have been optimized due to the acute consumption of the anti-inflammatory agent concurrent with exercise training. A study modelled off the Trappe et al. study could be initiated to determine the effects of blueberries on the muscle health of older adults. A 12-week resistance training program with 4 groups; low dose blueberry, high dose blueberry, ibuprofen, and placebo, could compare the effects of a natural food like blueberries to a pharmacologic agent like ibuprofen and ultimately determine the clinical implications of blueberry consumption’s effects on the skeletal muscle inflammation susceptibility of older adults.
Long-term, daily blueberry consumption does not appear to have any effect, positive or negative, on the inflammatory profile of aged skeletal muscle, nor on the inflammatory response 24 hours after eccentric exercise induced muscle damage. Further research on the acute effects of blueberry consumption might elucidate mechanisms of inflammatory modulation of aged muscle as seen with other foods and pharmacologic agents, but daily consumption itself does not appear to cause inherent muscle inflammatory changes.


Acknowledgements: The authors would like to thank all research volunteers for their contributions as well as the many student volunteers who contributed their time helping make this project possible. This work was supported by funding from the United States Highbush Blueberry Association (E.K. Merritt) and the Appalachian State University Graduate Research Assistant Mentorship Award (D.A. Arminavage).

Author Contributions: E.K.M., L.S.M., and S.R.M. conception and design of research. All authors performed experiments. C.D, L.B., J.M.B., D.A., V.G., E.K.M., L.S.M., and S.R.M. analyzed data and interpreted results of experiments. C.D., V.G., and E.K.M. prepared figures, C.D, and E.K.M. drafted manuscript, C.D., E.K.M., L.S.M., and S.R.M. edited and revised manuscript. All authors approved final version of manuscript.

Conflict of interest statement: No conflicts of interest, financial or otherwise, are declared by the authors.

Ethical standard: This study was reviewed and approved by the Appalachian State University Institutional Review Board and complies with all laws of the U.S.A.



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G.O. Gjevestad1,2, H. Hamarsland3, T. Raastad3, J.J. Christensen1,4, A.S. Biong2, S.M. Ulven1, K.B. Holven1,5


1. Department of Nutrition, Institute of Basic Medical Sciences, P.O. Box 1046, Blindern, 0317 University of Oslo, Norway; 2. TINE SA, Centre for Research and Development, P.O. Box 7, Kalbakken, 0902 Oslo, Norway; 3. Department of Physical Performance, Norwegian School of Sport Sciences, P.B. 4104 U.S., 0806 Oslo, Norway; 4. The Lipid Clinic, Oslo University Hospital Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo, Norway; 5. National Advisory Unit on Familial Hypercholesterolemia, Department of Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital, P.O. Box 4950 Nydalen, 0424 Oslo, Norway.

Corresponding Author: Kirsten B. Holven, Department of Nutrition, Institute of Basic Medical Sciences, P.O. Box 1046, Blindern, 0317 University of Oslo, Norway. k.b.holven@medisin.uio.no

J Aging Res Clin Practice 2017;6:182-190
Published online September 18, 2017, http://dx.doi.org/10.14283/jarcp.2017.24



Objective: To investigate the effects of eleven weeks of strength training combined with two isocaloric protein supplements on mRNA expression levels in skeletal muscle and peripheral blood mononuclear blood cells (PBMCs). Design: A double blind randomized controlled study. Setting: The Norwegian School of Sports Sciences, Norway. Participants: Untrained, but otherwise healthy, men and women (n=20, ≥ 70 yrs). Intervention: Participants were randomly allocated to receive either milk protein or a native whey protein supplement (20 g protein, morning and afternoon) combined with a standardized strength training protocol (6-10 RM, 1-3 sets, 3 times/week) for eleven weeks. Measurements: The mRNA expression levels of immune-related genes were measured before and after the intervention period, using RT-qPCR. Cytokines were measured using ELISA. Results: PBMC mRNA expression of interleukin (IL) 6, IL8, chemokine (C-C motif) ligand (CCL) 3, and nuclear receptor subfamily (NR) 1 group H (H) member 3 decreased significantly after the intervention period, whereas the mRNA expression of toll-like receptor (TLR2) increased. In skeletal muscle, the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) and PPARGC1B decreased significantly, whereas the mRNA expression of CCL2, CCL5, TLR2, TLR4 and hypoxia inducible factor 1 alpha subunit (HIF1A) increased significantly after the intervention. We found no significant differences in circulating C-reactive protein and IL6 after the intervention period. The consumption of whey and milk proteins had similar effects on mRNA expression levels after strength training in skeletal muscle as well as PBMCs. Conclusion: Eleven weeks of strength training and protein supplementation reduced the PBMC expression levels of genes involved in the immune system as well as in metabolism, underlining the close interaction between these processes. The upregulation of other immune-related genes observed in PBMCs as well as in skeletal muscle needs further investigations, but may be related to protein supplementation and training adaptations. Different protein supplementation (milk or native whey) did not differentially modulate mRNA expression after the intervention period.

Key words: PBMC, skeletal muscle, mRNA, resistance training, cytokines.

Abbreviations: BCAA, branched chain amino acids; CCL, chemokine (C-C motif) ligand; RCT, reverse cholesterol transport; CRP, C-reactive protein; CXCL, chemokine (C-X-C motif) ligand; E %, energy percent; HIF1A, hypoxia-inducible factor 1-alpha; HMBS, hydroxymethylbilane synthase; IL, interleukin; IL1RN, interleukin 1 receptor antagonist; IMVC, isometric maximum voluntary contraction; IPO8, importin 8; NR4A2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; nuclear receptor subfamily 4, group A, member 2; NR4A3; nuclear receptor subfamily 4, group A, member 3; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; PBMCs, peripheral blood mononuclear cells; PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PPARGC1B, peroxisome proliferator-activated receptor gamma coactivator 1-beta; RCT, reverse cholesterol transport; RM, repetition maximum; RT-qPCR, real-time quantitative polymerase chain reaction; TBP, TATA box binding protein; TG, triglycerides, TLDA, TaqMan Low-Density array; TLR, toll-like receptor; TNF, tumor necrosis factor alpha.




Diet and exercise are two of the most important lifestyle factors influencing healthy aging. Both are able to induce changes in metabolism and immune function (1, 2). They are therefore important targets for lifestyle interventions aiming at preventing the development of age-related diseases, including cardiovascular diseases, diabetes type 2, obesity (3) and sarcopenia (4).
Regular physical activity improves several of the underlying metabolic processes that are associated with adverse health effects and age-related diseases, such as insulin sensitivity, plasma triglycerides, blood pressure, and endothelial function (5). Regular strength training promotes muscle hypertrophy mainly, being particularly important in the prevention of age-associated loss of muscle mass and function associated with sarcopenia (6). Cross-sectional and large-scale cohort-studies have consistently shown an inverse association between physical activity and circulating markers of low-grade chronic inflammation (7). This anti-inflammatory effect is suggested to be one of the mechanisms underlying the protective effects observed on the development of chronic metabolic diseases by regular exercise (5), including sarcopenia (8). Because older adults often have a higher baseline level of circulating immune-related markers (9), long term regular exercise has been suggested as a tool to ameliorate the inflammatory status of older adults (10).
Components of the diet may exert anti-inflammatory effects and protect against chronic low-grade inflammation (2). In line with this, some epidemiological findings indicate that low-fat dairy products may reduce circulating levels of immune-related markers (11). However, others have found no association between intake of dairy products and circulating levels of immune-related markers, and conclusions from intervention studies have been conflicting (12). Although possible mechanisms underlying a potential anti-inflammatory effect of dairy products remains to be elucidated, several components in milk have been suggested to extert the anti-inflammatory effects, among them whey proteins (13).
The aims of the present study were to investigate the effects of eleven weeks of strength training combined with two isocaloric protein supplements (20 g protein morning and afternoon) on mRNA expression levels in skeletal muscle and peripheral blood mononuclear blood cells (PBMCs) in healthy older adults (>70 yrs).


Materials and methods

Study population and experimental design

Twenty-four older (≥70 yrs) untrained men and women were recruited to an eleven week lasting double-blind, randomized controlled study, which was conducted from August 2014 to December 2014 at The Norwegian School of Sport Sciences, Norway. All participants provided written informed consent, and we conducted the study according to the guidelines laid down in the Declaration of Helsinki. The Regional Committees for Medical and Health Research Ethics, Health Region South East, Norway, provided approval for all planned procedures involving human subjects (2014/834).
Subjects were randomized into one of two groups, receiving dairy supplements based on either native whey protein or regular milk protein for eleven weeks, on top of a high-load strength training regime. All tests were performed before and after the 11-week period. From the day before the test days, subjects followed a standardized diet based on body weight. In the morning of the test day, subjects were served a standardized breakfast, consisting of oatmeal, water, rapeseed oil and sugar (50 E % from carbohydrate, 8 E % from protein and 42 E % from fat). All subjects finished the breakfast within 20 minutes. PBMCs and skeletal muscle samples were collected 90 min after breakfast was served. We used Dual energy X-ray absorptiometry (Lunar iDXA, GE Healthcare, Buckinghamshire, United Kingdom) and enCORE Software (version 14.10.022, GE Lunar) to determine fat mass. Android fat mass was estimated based on algorithms in the enCORE Software. All subjects reported to be healthy, but one participant (4 %) had prescribed anticoagulants and six participants (25 %) took statins. Four subjects did not complete the study due to disease (two participants) or hurting joints (two participants).

Protein supplements

TINE SA (Oslo, Norway) provided the supplements. The products contained 20 g of protein (27 E %), 39 g carbohydrates (52 E %) and 7 g fat (21 E %), providing approximately 300 kcal per serving. All subjects received 2×20 g protein/day. The protein source was either native whey protein or regular milk protein (approximately 80 % casein and 20 % whey). Native whey proteins differ from regular whey proteins by the production method (produced at low temperatures to avoid extensive denaturation of the protein) and the composition of amino acids (e.g. higher amount of leucine). The participants prepared one serving in the morning and one in the evening, except at training days, where the training instructors provided the evening dose following the exercise sessions. The powder was dissolved in approximately 500 ml water. The producer delivered supplements in identical and coded packaging to ensure blinding of both the providers and the participants. The producer was responsible for coding of the products, and the coding was not revealed until the intervention was completed. All products had the same flavor, color and appearance.

Training protocol

The training program was standardized in terms of exercises performed (hammer squat, leg press, knee extensions, calf raise, chest press, seated rowing, close grip pull down, shoulder press, back extensions and crunches), the number of sets (6-12 repetition maximum, RM) and the number of repetitions (1-3 sets in each exercise). The exercise program was performed 3 days per week for eleven weeks (Figure 1), and all training sessions were supervised by trainers. The load in each exercise was individualized and adjusted each training session by the trainer to make sure that participants always exercised at the intended training load. Participants completed the last exercise session at least three days before the last biopsy was taken.

Figure 1 Study design and timeline for sampling during the intervention study

Figure 1
Study design and timeline for sampling during the intervention study


Sampling and sample preparation

We collected venous blood samples in BD Vacutainer® CPTTM cell preparation tubes with sodium heparin (Becton Dickinson, NJ, USA) and in silica gel tubes (Becton Dickenson Vacutainer Systems, Plymouth, UK). Within two hours of blood collection, PBMCs were isolated according to the manufacturer’s instructions (Becton Dickinson, NJ, USA). Serum samples were centrifuged (1500 g, 15 min at RT) after being left on the bench top for at least 30 min. Blood plasma samples were collected with pipettes immediately after being collected (lithium heparin tubes from Vacuette, Greiner Bio One, Austria) and centrifuged (1300 g, 10 min at 4°C). All samples were stored at -80°C until further analysis.
Muscle biopsies from the m. vastus lateralis were obtained at the same time points as the blood samples, using a modified Bergstrom technique (14), immediately cleaned from blood and connective tissue in physiological salt water at 4˚C, immersed into RNAlater® solution (Ambion, TX, USA) and stored overnight at 4 ˚C. The following day, the biopsies were transferred and stored at – 80 ˚C until further analysis. Biopsies were taken from the same leg before and after the intervention.

Isolation of RNA and synthesis of cDNA

Skeletal muscle samples were ruptured using a CryoGrinder System (Ops Diognostics, NJ, USA), followed by homogenization in Quizol (QIAGEN GmbH, Germany) and addition of chloroform. After centrifugation, the upper phase was transferred to a fresh tube and ethanol added. The procedure of RNA isolation was carried out using the QiaCube instrument (QIAGEN GmbH, Germany) following the miRNease Mini Kit protocol (QIAGEN). We isolated RNA from PBMCs in accordance with the RNeasy Mini Kit protocol using qiashredder and DNase digest (QIAGEN GmbH, Germany) using the QiaCube. High-quality RNA from both PBMCs and skeletal muscle samples was eluted in 30 μl of RNase free water and frozen at -80 °C until further analysis. RNA quantity was measured using NanoDrop-1000 (NanoDrop Technologies, Inc., DE, USA), and RNA quality was checked with Aglient 2100 Bioanalyzer (Agilent Technologies, Inc., CA, USA). All samples included in further analysis had a RIN-value above 5.5. PBMC samples from one person were not taken due to a misunderstanding in the laboratory, whereas mRNA from skeletal muscle was lost from six participants due to low RIN-values. In addition, we excluded some of the mRNA transcripts from our final analysis due to abnormal multicomponent plots.
RNA samples were transcribed into cDNA (500 ng) using the cDNA kit from Applied Biosystems (Applied Biosystems, UK) and in accordance with the protocol provided. Samples were stored at -20 °C for further analysis.

RNA analysis by real-time qPCR

We measured mRNA levels of 48 genes (Additional file 1) using TaqMan Low-Density array (TLDA) cards from Applied Biosystems (UK). The TLDA cards were used on a 7900 HT Applied Biosystems RT-qPCR machine (Applied Biosystems, UK). The Ct-values were analysed using SDS 2.4 (Applied Biosystems, UK), and further transferred to ExpressionSuite Sofware v1.0.3 (Applied Biosystems, UK). For PBMCs, we normalised the Ct-values to TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) mRNA transcripts, whereas for skeletal muscle, we normalized to TBP and importin 8 (IPO8). Fold changes in mRNA transcripts from baseline to end of study were calculated, using the 2-ΔΔCt-method (15).
The selection of genes were based on a thorough literature search investigating the effect of training on gene expression in PBMCs (16). We performed a similar research for skeletal muscle (17). Moreover, the genes selected in the present study were based on previously published studies where the effects of dairy products on markers of chronic low-grade inflammation were described (18).

Cytokine measurements and routine analysis

The serum level of IL6 was measured with high-sensitive Quantikine ELISA (R&D Systems Inc., MN, USA), whereas IL8 and CCL3 were measured using Quantikine ELISA (R&D Systems Inc.), both in accordance with the protocols provided. We measured all samples in duplicates. Serum levels of glucose, triglycerides, cholesterol and the plasma level of C-reactive protein (CRP) were analyzed by an accredited laboratory (Fürst Medical Laboratory, Oslo; Norway).


Power calculation was performed for the primary outcome of the study, which was to study the effects of consuming native whey or milk protein on muscle mass and strength. We also considered this number of participants to be relevant with respect to changes in inflammatory markers. In addition, the number of participants included in the present study is in line with other studies exploring the relationships between exercise and gene expression . All data were checked for normality. Subjects with levels of CRP above >10 mg/L at baseline (n=1) or at end of study (n=1) were excluded from the gene expression analysis as such levels may indicate an ongoing acute inflammation, not reflecting the intervention. For non-parametric data, we used the Mann-Whitney-test for independent measurements and the Wilcoxon signed-rank for repeated, paired measurements. For parametric data, Independent samples t-test was used for independent measurements and Paired t-test for paired measurements. The Spearman correlation test was used to reveal possible correlations between the change in android fat mass and mRNA expression levels of selected transcripts. Due to an explorative study design, correction for multiple testing was not performed. We considered a p-value of < 0.05 statistically significant. SPSS statistical software, version 22 from Microsoft (SPSS, Inc., CA, USA), was used for statistical calculations and GraphPad Prism 5 (GraphPad Software, Inc., CA, USA) for generating figures.



Participants (n=20, mean ± SD=73.6 ± 2.8 yrs) included in the present study were similar in body mass, fat mass, BMI and blood parameters at baseline. The proportion of male to female was 12/8. We observed a significant change in body mass and BMI from baseline to end of intervention, but not in fat mass or android fat mass (Table 1). None of the anthropometric characteristics or blood parameters changed significantly from baseline to end of study between the native whey and the milk group (data not shown).


Table 1 Anthropometric parameters at baseline and after the intervention

Table 1
Anthropometric parameters at baseline and after the intervention

1. Calculated by Paired sample t-test; 2. n=19; Abbreviations used in table; Δ, delta; HDL, high-density cholesterol; LDL, low-density cholesterol

Adherence to the strength training and protein supplementation

Participants attended an average of 33.0 ± 0.9 and 32.5 ± 1.2 exercise sessions (of totally 33 exercise sessions) in the native whey and milk group, respectively. We logged adherence to the supplementation regime at each training session, which resulted in a mean self-reported compliance of 99 %.

Effects of strength training and protein supplementation on gene expression

Protein supplements based on milk protein or native whey protein did not significantly alter mRNA expression levels after eleven weeks of strength training neither in PBMCs nor in skeletal muscle. When merging the two groups, PBMC mRNA expression levels of interleukin (IL) 6, IL8, chemokine (C-C motif) ligand (CCL) 3 and nuclear receptor subfamily 1, group A, member 3 (NR1H3, also known as LXR) were significantly reduced after the intervention period, whereas the mRNA expression level of toll-like receptor (TLR) 2 increased (Figure 2). In skeletal muscle, the mRNA expression levels of CCL2, CCL5, TLR2, TLR4, IL8 and hypoxia-inducible factor 1-alpha (HIF1A) significantly increased after the intervention (Figure 3 and 4), whereas peroxisome proliferator-activated receptor gamma coactivator -alpha (PPARCG1A) and PPARCG1B decreased significantly (Figure 4). We also observed significant changes in the expression of some genes related to lipid metabolism, both in PMBCs and skeletal muscle, as shown in Additional file 2 and 3.

Figure 2 mRNA expression levels in PBMCs before and after the intervention

Figure 2
mRNA expression levels in PBMCs before and after the intervention

Expression levels of IL6 [A], IL8 [B], CCL3 [C], NR1H3 [D] and TLR2 [E] from baseline to 11 weeks of strength exercise. Values are expressed as fold changes, and the vertical lines represent median values. The p-values were calculated by Wilcoxon signed-rank test and indicate changes from baseline til end of study. n = 14 [D], n= 15 [B, C] and n= 16 [A, G].

Figure 3 mRNA expression levels in skeletal muscle before and after the intervention

Figure 3
mRNA expression levels in skeletal muscle before and after the intervention

Expression levels of CCL2 [A], CCL5 [B], TLR2 [C], TLR4 [D] and IL8 [E] from baseline to 11 weeks of strength exercise. Values are expressed as fold changes and the vertical lines represent median values. The p-values were calculated by Wilcoxon signed-rank test and indicate changes from baseline til end of study. n = 10 [B, E], n = 11 [A] and n= 12 [C, D].

Figure 4 mRNA expression levels in skeletal muscle before and after the intervention

Figure 4
mRNA expression levels in skeletal muscle before and after the intervention

Expression levels of PPARGC1A [A], and PPARGC1B [B] and HIF1A [C] from baseline to 11 weeks of strength exercise. Values are expressed as fold changes and the vertical lines represent median values. The p-values were calculated by Wilcoxon signed-rank test and indicate changes from baseline til end of study. n = 12.

Circulating immune-related markers

No differences were observed from baseline to after the intervention in the serum level of IL6 or the plasma level of CRP (Table 2). Serum levels of IL8 and CCL3 were also measured, but were not detectable (not shown).


Table 2 Inflammatory markers at baseline and after the intervention

Table 2
Inflammatory markers at baseline and after the intervention

1. Calculated by Wilcoxon signed rank test; 2. n=14; Abbreviations used in table; CRP, C-reactive protein; Δ, delta; IL, interleukin.

Correlations between android fat mass and immune-related genes and circulating markers

No correlations between changes in android fat mass and changes in mRNA expression levels of IL6, IL8 and CCL3 in PBMCs or circulating levels of IL6 and CRP were observed from baseline to end of intervention.



We observed that 11 weeks of high-load strength training combined with protein supplementation decreased mRNA expression levels of several immune-related genes in PBMCs, potentially having beneficial effects on systemic low-grade inflammation. On the other hand, immune-related mRNA transcripts were both up- and downregulated in skeletal muscle, probably reflecting muscle regeneration and adaptation. Native whey and milk proteins did not differentially alter mRNA expression levels, neither in PBMCs nor in skeletal muscle after strength training. No effects were observed on circulating levels of IL6 or CRP.

It is well known that long-term adaptations to strength training result in increased muscle mass (hypertrophy) and strength (6). Furthermore, regular exercise may reduce the level of immune-related markers (1). This is in line with observations from the present study, as we found reduced mRNA expression levels of IL6, IL8 and CCL3 in PBMCs after the training period. It is also in line with a study where PBMC mRNA expression levels of immune-related markers of middle-aged men and women (n=40, mean age 50-67 yrs) were reduced (CCL2), or tended to be reduced (TNF) after two months of brisk walking (6 days/week, 50 min/day, 70% of maximal heart rate). However, the mRNA expression level of IL6 did not change after the training period in that study (10). Importantly, the older subjects had higher baseline levels of the relevant markers compared to younger participants. The authors therefore suggested that the observed reduction might be related to the higher baseline levels of these mRNA transcripts in the older compared to the younger participants, and that these differences promoted a more robust reduction after the exercise period in older adults (10).
In contrast to the decreased mRNA levels of IL6, IL8 and CCL3 in PBMCs, we observed an increased mRNA expression level of TLR2 in PBMCs as well as in skeletal muscle. The mRNA expression of TLR4, IL8, CCL2 and CCL5 were also significantly increased in skeletal muscle after the intervention. These results are in contrast to others who have found decreased mRNA expression levels of TLRs in PBMCs (19), skeletal muscle (20) and whole blood (21) after regular strength training in older adults. Increased levels of TLRs may induce NF-κB activation in PBMCs as well as skeletal muscle and contracting C2C12 myotubes have been shown to induce CCL2 in an NF-κB-dependent manner (22). In skeletal muscle, it has been shown that NF-κB activation may prevent myogenic differentiation (23) and contribute to muscle atrophy by increasing the activity of molecules involved in muscle protein degradation (24). However, this seems unlikely to occur in the present situation as we observed increased muscle mass after the training period and reduced levels of IL6, IL8 and CCL3 in PBMCs. Similarly, increased NF-κB activation in PBMCs is closely linked to the development of low-grade inflammation (25), but NF-κB inhibition during the resolution phase can also prevent proper tissue repair (26). IL8, CCL2 and CCL5 are chemoattractants, which may play an important role in the recruitment of immune-related cells to skeletal muscle following an acute exercise bout (27). Long-term effects of training on these markers are less investigated, but circulating levels of IL8 and CCL2 have been shown to decrease after aerobic training programs (28). The mission of immune-related markers in exercise is not fully understood, but immune-related markers are hypothesized to be important in the resolution processes by removing cellular debris, releasing factors to promote muscle growth and to facilitate vascular and muscle fibre repair, amongst others (29). TLRs may potentially also activate other pathways, such as p38 mitogen-activated kinase (MAPK) and C-Jun N-terminal kinase (JNK) (30)possibly stimulating cell proliferation (31).
Furthermore, we observed decreased mRNA expression levels of NR1H3 in PBMCs, and of PPARGC1A and PPARGC1B in skeletal muscle after the training period. In contrast to these results, regular endurance training has been shown to upregulate the expression of NR1H3 in PBMCs (32) and PPARGC1A and PPARGC1B in skeletal muscle (33). However, a decreased mRNA expression level of PPARGC1B has also been observed in skeletal muscle after 10 weeks of knee extensor training in young adults (n=7, mean age 26±1 yrs) (34). NR1H3 plays a central role in the transcriptional regulation of both cholesterol homeostasis and inflammation (32, 35). The signaling pathway inducing NR1H3 expression in macrophages involves NF-κB dependent transcriptional gene activation and may promote anti-inflammatory effects (35). Further, TLRs have been shown to downregulate NR1H3 (35), which we also observed in PBMCs in the present study. PPARGC1A and HIF1A are thought to be involved in energy metabolism (36), and PPARGC1A is also closely linked to inflammation (37). Lower levels of PPARGC1A have been observed in patients with diabetes type 2 [38], the metabolic syndrome (39) and in aging (38). A possible explanation for the conflicting results of NR1H3, PPARGC1A and PPARGC1B in the present and other studies may be that participants in the present study also consumed protein supplements, which potentially may alter mRNA expression levels of genes investigated.

In contrast to the observed decrease in the mRNA expression level of IL6 in PBMCs in the present study, the circulating level of IL6 was unchanged. This discrepancy support the notion that PBMCs are not the main source of circulating IL6 (40). Further, a stable level of circulating IL6 has previously been found after five consecutive days of high-volume resistance training (41) and after three months of combined endurance and resistance training (42). At the same time, reduced levels of circulating IL6 have been observed after progressive 24-wk exercise of endurance exercise (43) and after walking 10000 steps three times per week for eight weeks (44). Furthermore, we found no changes in the level of CRP after the intervention period. This is supported by a recent study where community dwelling older adults performed a strength training program for 12 weeks (45), but is in contrast to the findings in a combined aerobic and resistance training program in middle-aged men and women where the level of CRP was reduced after 6 months of training (46). Strength training has also been shown to decrease CRP in overweight women (47) and in older adults (48).
Possible mechanisms underlying the anti-inflammatory effects of training are largely unknown, but it has been hypothesized that the reduced levels of immune-related markers observed after training are due to a redistribution of fat mass (1). In the present study, we did not find any significant changes in the distribution of android fat mass and we observed no correlations between changes in mRNA transcripts of IL6, IL8 and CCL3 and android fat mass after the intervention period. Despite no changes in android fat mass, the mRNA expression levels of IL6, IL8 and CCL3 in PBMCs decreased, indicating that the change in PBMCs may occur in the absence of a changed fat distribution.
Protein supplementation, in combination with strength training, may additionally enhance muscle protein synthesis and muscle hypertrophy (49). In addition, whey proteins are hypothesized to exert anti-inflammatory effects (50). CRP was reduced in elderly adults with sarcopenia (n=130, mean age 80.3 yrs) after 12 weeks of supplementation with whey protein (22 g), essential amino acids (10.9 g, including 4 g leucine) and vitamin D (2.5 µg) concurrent with a combination of regular endurance and strength training (51). Moreover, intake of branched chain amino acids (BCAA) has been shown to attenuate inflammation after a three-day extensive aerobic training program compared to an isocaloric amount of carbohydrate (52). However, no change was observed in serum concentrations of IL6 after resistance training combined with protein and omega-3 supplementation in novice resistance trained females (n=28, mean age 20±1 yrs) (41), and a recent meta-analysis showed that whey supplements do not modulate inflammation in healthy adults (53). Nevertheless, in the same meta-analysis, whey supplements lowered serum levels of CRP in subjects with initial high baseline values (53) such as in older adults (9), potentially providing a greater reduction of inflammatory markers in these groups compared to groups with lower baseline levels (54). However, our results do not support this notion, as we observed no differences in circulating levels of CRP or IL6 between the whey and the milk group after the intervention period (results not shown).
Most training studies have been exploring the effects of aerobic exercise on gene expression levels, both in PBMCs and skeletal muscle. Whereas adaptations to endurance training include mitochondrial biogenesis and enhanced aerobic metabolism (55), strength training mainly promotes hypertrophy (56). Different types of exercise are therefore likely to induce different subsets of genes, possibly explaining some of the contradictory observations in the present and previous studies. The fact that subjects in the present study consumed protein supplements combined with the exercise intervention may also have affected mRNA expression levels of selected genes. Further, the differences we observed in mRNA expression levels in PBMCs and in skeletal muscle after eleven weeks of strength training may reflect the different functions and adaptation processes to regular exercise in these two tissues. Differences in initial health status, exercise intensity (57), duration of the exercise performed and sampling points (58) may also explain the conflicting results in PBMCs (16), serum (59) and skeletal muscle (20). The frequency and exercise load may ultimately determine whether the organism responds with favorable adaptations, or, in the case of inadequate recovery, experiences increased inflammation (60). Further, the results from the present study underline the complex regulation and integration of metabolism and inflammation (61) and show that a combined exercise and supplementation intervention were able to alter the expression of genes involved in energy metabolism and inflammation in PBMCs as well as skeletal muscle. The physiological importance of the observed effects of strength training and protein supplementation on immune-related markers in PBMCs, as well as skeletal muscle is unclear and needs further investigations.
Major strengths to the present study are the randomized controlled design, with participants receiving a standardized diet prior to the sampling. All exercise sessions were standardized and performed under close supervision, and blood samples and muscle biopsies were collected simultaneously allowing us to compare the responses in two tissues. There are also some limitations to the present study. Few subjects were included in the study, and the lack of non-significant results may be due to the low number of participants. Further, no adjustments for, or comparison between, gender was performed. We did not include a non-training control group, or a group who did not consume the protein supplements. This makes us unable to conclude that the training intervention was the sole cause of the altered gene expression level observed or to distinguish the effects between the training and the supplementation. The observed changes in some of the inflammatory markers may therefore be related to changes in the diet and may potentially explain the conflicting results between the present intervention study and intervention studies were training is the only intervention.



In the present study, we found reduced levels of some immune-related markers in PBMCs after eleven weeks of high-load strength training, possibly providing protection against chronically related diseases, such as atherosclerosis. Simultaneously, we observed increased levels of other immune-related mRNA transcripts in PBMCs as well as in skeletal muscle. These changes may reflect muscle regeneration and adaptation. However, we need further investigations of the physiological impact of these changes in PBMCs as well as in skeletal muscle. Further, we observed no differences in mRNA expression levels between participants consuming native whey proteins compared to those consuming regular milk proteins during the training period. The present study emphasizes that a combined training and supplementation intervention exert both local (muscle tissue) and systemic effects (PBMCs) and that diet may interfere with this response. However, due to the study design we were not able to separate the effects of the supplementation from the effects of the training.


Acknowledgements: We want to acknowledge all the participants volunteering to the study.

Competing interests: The work was supported by The Research Council of Norway (project number 225258/E40), Throne Holst Foundation for Nutrition Research (University of Oslo), The Norwegian School of Sports Sciences (NIH) and TINE SA.

Conflict of interest: I.O., T.R., H.H., J.J.C., K.B.H. and S.M.U. report no conflict of interest. The test products were provided by TINE SA, Oslo, Norway, where G.O.G. and A.S.B. are researchers employed, G.O.G. as an industrial PhD-student. They have no financial interest to declare. K.B.H. has received research grant from TINE SA, Mills DA, Olympic Seafood, Amgen, Sanofi and Pronova. S.M.U. has received research grant from TINE SA, Mills DA and Olympic Seafood. T.R. has received grants from TINE SA.

Ethic approval: The study complies with the current laws in Norway.

Authors’ contributions: Conception or design of the study; T.R., H.H., A.S.B., S.M.U. and K.B.H. Acquisition, analysis or interpretation of the work; all authors. Drafting or critically revising the manuscript; all authors. Read and approved the final manuscript; all authors.



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