Endocrine DOI 10.1007/s12020-014-0501-x
ORIGINAL ARTICLE
Time course of IL-15 expression after acute resistance exercise in trained rats: effect of diabetes and skeletal muscle phenotype Mahdieh Molanouri Shamsi • Zuhair Mohammad Hassan LeBris S. Quinn • Reza Gharakhanlou • Leila Baghersad • Mehdi Mahdavi
•
Received: 14 September 2014 / Accepted: 3 December 2014 Ó Springer Science+Business Media New York 2014
Abstract Type 1 diabetes is associated with skeletal muscle atrophy. Skeletal muscle is an endocrine organ producing myokines such as interleukin-15 (IL-15) and interleukin-6 (IL-6) in response to contraction. These factors may mediate the effects of exercise on skeletal muscle metabolism and anabolic pathways. Lack of correlation between muscle IL-15 mRNA and protein levels after exercise training has been observed, while regulatory effects of IL-6 on IL-15 expression have also been suggested. This study determined post-exercise changes in muscle IL-15 and IL-6 mRNA expression and IL-15 protein levels in healthy and streptozotocin-induced diabetic rats in both the fast flexor hallucis longus (FHL) and slow soleus muscles. Resistance training preserved FHL muscle weight in diabetic rats and increased IL-15 protein levels in both the soleus and FHL muscles. However, the temporal pattern of this response was distinct in normal and diabetic rats. Moreover, discordance between post-exercise muscle IL-15 mRNA and protein expression was observed in
our study, and diabetes suppressed post-exercise increases in FHL muscle IL-6 mRNA expression. Our study indicates that training, skeletal muscle phenotype, and metabolic status all influence the temporal pattern of post-exercise changes in IL15 expression. Muscle IL-15 protein levels increase following training, suggesting this may be an adaptation contributing to increased capacity for secretion of this myokine that is not depressed by the diabetic state.
M. Molanouri Shamsi (&) R. Gharakhanlou L. Baghersad Physical Education & Sport Sciences Department, Faculty of Humanities, Tarbiat Modares University, Tehran, Islamic Republic of Iran e-mail: [email protected]
Z. M. Hassan Jala Ale Ahmad Exp., P.O. Box 14115-13116, Tehran, Islamic Republic of Iran
R. Gharakhanlou e-mail: [email protected] L. Baghersad e-mail: [email protected] M. Molanouri Shamsi R. Gharakhanlou L. Baghersad Jala Ale Ahmad Exp., P.O. Box 14117-13116, Tehran, Islamic Republic of Iran
Keywords Interleukin-15 Interleukin-6 Resistance training Type-1 diabetes Exercise Myokines Cytokines Introduction Skeletal muscle atrophy is considerable in type 1 diabetes [1, 2]. In healthy adults, skeletal muscle displays a remarkable capacity for growth, adaptation, and regeneration in response
L. S. Quinn Division of Gerontology and Geriatric Medicine, Department of Medicine, Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System, University of Washington, Seattle, WA 98108, USA e-mail: [email protected] M. Mahdavi (&) Immunology Department, Pasteur Institute of Iran, 69 Pasteur Ave, Tehran, Iran e-mail: [email protected]
Z. M. Hassan Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran e-mail: [email protected]
123
Endocrine
to exercise and changes in load [3]. Skeletal muscle produces myokines in response to contraction that may thereby provide a feedback loop for the muscle to regulate its own growth and regeneration, allowing for adaptation to exercise training [4, 5]. Interleukin-15 (IL-15) is a recently discovered myokine that is highly expressed in skeletal muscle tissue [6, 7]. It has been shown that IL-15 can affect body composition independent of immune cells [8]. In addition to its anabolic effects on skeletal muscle, it has been suggested that IL-15 may modulate insulin sensitivity in skeletal muscle [9–11]. Recently, we have shown that diabetes modulates the effects of exercise on IL-15 expression in skeletal muscle [12]. While skeletal muscle IL-15 protein levels increased after chronic exercise training in diabetic rats, comparable changes were not observed for IL-15 mRNA expression, which may be attributed to the complex regulation of IL-15 transcription and translation [7, 12, 13]. IL-6 has also been suggested to function as an exercise-related myokine that has metabolic and hypertrophic actions in skeletal muscle [14]. IL-6 transcription and mRNA expression are markedly upregulated in skeletal muscle during exercise [15], and IL-6 appears to signal through autocrine, paracrine, and hormonal mechanisms to modulate muscle mass and metabolism [5, 16]. Regulatory effects of IL-6 on IL-15 expression have been observed in some studies [17, 18]. Resistance-type exercise training has been shown to represent an effective interventional strategy to augment muscle mass, strength, and function in diabetes [12, 19, 20]. Skeletal muscle tissue is sensitive to the acute and chronic stresses associated with resistance training. These responses are influenced by the structure of resistance activity as well as the training history of the individuals involved. At present, there is conflicting evidence about the exercise-induced changes of IL-15 mRNA and protein in skeletal muscle by training. Some of the disparities reported in the literature may be due to differences in post-exercise muscle sampling intervals, which range from immediate to 24 h post-exercise [12, 21, 22]. In this study, we hypothesized that post-exercise changes in muscle IL-15 mRNA versus IL-15 protein levels would be discordant, and further would be temporally distinct in normal and diabetic rats. Our findings indicate that diabetes, training status, and skeletal muscle type can all modulate post-exercise changes in myokine expression, but that the training-induced increase in muscle IL-15 content is preserved in diabetes.
other scientific purposes, and the protocol was approved by the Ethics Committee of the School of Medicine Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Male Wistar rats weighing 250–280 g were used in this study and were housed in a light- and temperature-controlled animal facility with food and water provided ad lib. Animals were maintained in the Central Animal House, School of Medical Sciences of TMU. Experimental design Animals were randomly assigned to the following treatment groups: control (C, 12 per group); trained (T, 16 per group); STZ-induced diabetes (D, 12 per group); and STZinduced diabetes plus training (DT, 16 per group). Diabetes was induced with IP injection of STZ (Sigma) at 55 mg kg-1 of body weight (BW) in a 0.1 M citrate buffer (pH 4.5). An equal volume of buffer was injected into the control rats. Blood glucose concentrations were assessed after 4 days to ensure that fasting levels greater than 14 mmol l-1 (250 mg dl-1) were reached. Diabetic rats were not treated with insulin during the study, and they showed symptoms of type1 diabetes, such as polyuria and weight loss. Rats in the T and DT groups were trained using a ladder-climbing protocol with progressively larger weight loads attached to the tail, as previously described [23]. Briefly, animal climbed 26 rungs across a 1-m ladder. One repetition along the ladder required 26 total lifts by the animal (13 lifts per limb). Rats were familiarized with the exercise for 3 days, 48 h before STZ injection, and exercise training was initiated after STZ injection. Rats were positioned at the bottom of climbing apparatus and motivated to climb the ladder by touching the tail. Animals were given exercise training with a rest of 48 h between each exercise session. Animals from the T and DT groups were exercised with five sets of four repetitions each with a 60-s rest interval between the reps and 3 min between the sets per session. At 13 and 14 sessions, rats were decreased to 3 sets of 5 repetitions. Weight loads were based on pilot studies for T and DT groups and previously described [12, 20] and are provided in Fig. 1. Based on our pilot studies and previous literature reports [12, 20, 21], it was not possible to use the same loads for healthy and diabetic rats. Tissue preparation
Methods Animals All experiments involving animals were conducted according to the policies of the Iranian convention for the protection of vertebrate animals used for experimental and
123
On the day of sacrifice, rats were randomly divided into four subgroups, C, T, D, and DT groups which, respectively, were sacrificed immediately, 4, 8, and 12 h following the last session of resistance training. Rats were anesthetized with a mixture of Ketamine (30–50 mg kg-1 BW, IP) and Xylazine (3–5 mg kg-1 BW, IP). The soleus and flexor hallucis longus (FHL) muscles, used as
Endocrine 160 140
Percent of Body Mass
expression levels of IL-15 and IL-6 were normalized by subtracting the corresponding levels of the mean of GAPDH and RPL-26 DCT, which were amplified as housekeeping genes. Data are represented as fold change from the C group [24].
Diabetic Nondiabetic
120 100 80
Muscle preparation
60 40 20 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17
Sessions Fig. 1 Protocol for resistance training using ladder climbing. Amount of weight lifted by diabetic and normal rats during 17 sessions of resistance exercise
representative fast-twitch and slow-twitch muscles, respectively, were quickly extracted, weighed, and stored in liquid N2 for subsequent analysis.
Frozen tissues (0.1–0.3 g) were homogenized in RIPA buffer (0.625 % Nonidet P-40, 0.625 % sodium deoxycholate, 6.25 mM sodium phosphate, and 1 mM EDTA at pH 7.4) containing 10 lg ml-1 of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri). Homogenates were centrifuged at 12,000 g for 10 min at 4 °C, the supernatant was saved, and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, California) with bovine serum albumin as a Ref. [25]. Supernatants were aliquotted and stored at -80 °C. Assay of IL-15 protein in skeletal muscles
Real-time PCR Total RNA was extracted from frozen soleus and FHL muscles samples using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s recommendations. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity was determined by calculating the absorbance ratio at 260 and 280 nm and by ethidium bromide staining. Purification was accepted when the 260/280 nm absorbance ratio was above 1.8. Isolated RNA was stored at -80 °C. RNA was reverse transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent Kit (Perfect Real Time, Takara Code RR037A, Japan) using the following protocol: reverse transcription at 37 °C for 15 min, inactivation of reverse transcriptase at 85 °C for 5 s, and refrigeration at 4 °C for 10 min, with storage at -20 °C. Primer sets for rat GAPDH (glyceraldehyde 3-phosphate dehydrogenase), RPL-26 (ribosomal protein L26), IL-15, and IL-6 were produced by Qiagen QuantiTect (GAPDH, QT00199633; RPL-26, QT01828771; IL-15, QT01813637 and IL-6, QT00182896). Gene expression was measured by real-time PCR using the Rotor-Gene 6000 (Corbett Research, Mortlake, Australia). The thermal cycle protocol was as follows: 1 cycle at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. PCR amplification was performed in duplicate in a total reaction volume of 20 ll. The reaction mixture consisted of 3 ll diluted template, 10 ll SYBR Premix Ex TaqTMKit (Perfect Real Time, Takara Code RR041A, Japan), and 2 ll primers. Amplification specificity was controlled by a melting curve analysis and gel electrophoresis of the PCR products. Relative
IL-15 protein concentrations in muscle were determined in duplicate using the USCN ELISA kit (USCN Life Science Inc., Wuhan,China or Houston, TX). The assays were carried out according to the manufacturers’ instructions. Muscle IL-15 data are expressed as pg of cytokine per mg of total protein. The minimum detectable concentration was \5.6 pg ml-1 for IL-15. The intra- and inter-assay coefficients of variation were 10 and 12 % for IL-15, respectively. Blood glucose and serum insulin measurements Fasting blood was sampled from the tail vein in rats before sacrifice. Blood glucose levels were tested by glucometer (GT-1920, Japan) with samples run in duplicate. Serum insulin concentrations were measured using a commercially available ultrasensitive rat insulin ELISA kit (ALPCO Diagnostics, Windham, NH). Statistical analysis All analyses were performed using SPSS V16.0 (SPSS, Chicago, IL). Three-way analysis of variance (three-way ANOVA) and Tukey post hoc tests were used for phenotypic, mRNA, and protein data in different time course following resistance exercise. Also, one-way ANOVA and Tukey post doc tests were used for mRNA and protein data in different groups per time course following resistance exercise. Statistical significance was set at P \ 0.05. Data are presented as mean ± SEM.
123
123
50.8 ± 2.3a
Within rows, values with different letters are different at P \ 0.05; values with the same letter are not significantly different
48.4 ± 1.8a 49.3 ± 2.4a 51.7 ± 2.6a 47.8 ± 1.5a
IW initial weight (g), FW final weight (g), FG fasting glucose (mmol l ), FI fasting insulin (ng ml ), FMM flexor hallucis longus (FHL), muscle mass (mg), FMBR FHL-to-body mass ratio (mg g-1 9 100), SMM soleus muscle mass (mg), SMBR soleus-to-body mass ratio (mg g-1 9 100)
-1
50.06 ± 3.5a 52.1 ± 1.7a 46.6 ± 3.7a SMBR
47.8 ± 2.8a
664.5 ± 7.8b
4.5 ± 0.24a
211.5 ± 6.5b 159.7 ± 5.7a 212.1 ± 3.5b 155.2 ± 8.7a
659.7 ± 12.3b 663.5 ± 15.3b
213.3 ± 5.4b 163.2 ± 11.4a 207.4 ± 7.6b 157.2 ± 16.9a 173.2 ± 3.7a 160.4 ± 14.3a FMBR SMM
676.2 ± 39.3b 556.6 ± 22.6a FMM
0.65 ± 0.05a
4.4 ± 0.1a 4.7 ± 0.1a
0.78 ± 0.06a 0.7 ± 0.07a
4.6 ± 0.1a
0.6 ± 0.04a
FG
FI
4.7 ± 0.04a
0.6 ± 0.03a
-1
46.06 ± 1.1a
199.4 ± 7.3b 118.7 ± 7.1b
481.5 ± 8.6d 477.5 ± 8.2d
207.3 ± 8.8b 114.5 ± 9.2b 202 ± 7.3b 121.7 ± 7.8b
471 ± 10.7d 474 ± 4.3d
201.1 ± 5.2b 113 ± 4.6b 168.2 ± 7.4a 110.4 ± 3.02b
400.8 ± 11.8c
27.1 ± 1.5b
0.16 ± 0.02b 0.18 ± 0.05b
26.8 ± 1.6b 28.2 ± 2.8b
0.21 ± 0.02b 0.19 ± 0.04b
27.2 ± 2.1b 30.1 ± 1.1b
261 ± 9.2a 266 ± 7.1a
232 ± 13.3b 235.7 ± 13.7b
262.5 ± 6.2a 267.5 ± 10.5a
236.2 ± 8. 6b 315.7 ± 12.9a
263.7 ± 8a 265.7 ± 8a
311.5 ± 10.8a 311.7 ± 11.8a
265.5 ± 8a 269 ± 9.4a
328.2 ± 27.7a
267.1 ± 5.3a
320.3 ± 7.5a
IW
FW
T12 T4 T0 C
Table 1 Characteristic features of rats in different groups
T8
IL-15 mRNA and protein expression in FHL and soleus muscles To determine the effects of training, diabetes, and time course on IL-15 mRNA and protein expression in slowand fast-twitch skeletal muscles, diabetic and normal rats were trained for 5 weeks with ladder climbing and sacrificed at different time points up to 12 h following the last session of resistance training. In the soleus muscle, a significant effect of diabetes, but not training or time, on IL15 mRNA expression was observed (Fig. 2a). Furthermore, we did not observe significant time-dependent differences within each of the four treatment groups for the soleus muscle (P [ 0.05), while immediately following exercise, IL-15 mRNA expression was significantly decreased in the DT group compared to controls (Fig. 2a). IL-15 mRNA expression in the fast FHL muscle exhibited a significant effect of diabetes, training, and time course, with the training-induced increase in IL-15 mRNA expression at 4 h blunted in the DT group (Fig. 2b). In contrast to the results for IL-15 mRNA expression, IL-15 protein levels in both FHL and soleus muscle showed significant effects of diabetes, training, and time. (Figure 2 c, d). Overall, IL-15 protein levels in both the
0.17 ± 0.03b
D
DT0
DT4
DT8
Rats underwent the protocols for controls (C0, C4, C8, and C12), resistance training (T0, T4, T8, and T12), STZinduced diabetes (D0, D4, D8, and D12) and diabetes with resistance training (DT0, DT4, DT8, and DT12) as outlined in Methods. The characteristic features of the rats in different groups for the first study are presented in Table 1, which shows that the diabetic groups displayed significant reductions in plasma insulin levels compared to the healthy groups. Consequently, typical type 1 diabetes hyperglycemia occurred in the diabetic groups, with the increase in fasting glucose. Moreover, at the end of the study, diabetic rats showed a significant decrease in total BW, although different groups did not have noticeable differences in total weight at the beginning of the study (Table 1). Mass of both the soleus and FHL muscles was lower in diabetic animals compared to controls (Table 1). Five weeks of resistance training resulted in hypertrophy of the fast FHL muscle in both normal and diabetic training groups, as indicated by a significantly higher FHL mass and FHL mass to body mass ratio (FMBR) in the trained animals compared to the sedentary animals (Table 1). Soleus muscles did not undergo hypertrophy in response to resistance training (Table 1).
263.2 ± 5.5a
DT12
Induction of diabetes mellitus and effects of training on muscle mass
241.4 ± 1b
Results
242.7 ± 12.7b
Endocrine
Endocrine
a
a
3
a
2.5
a
a
2 1.5
Training:P=0.93 Diabetes: P=0.001 Time Course:P=0.1 Intraction: P=0.46
a
*
1
a a
a
a
*
a
a
a
a
C
a a
T D DT
0.5 0
Ex + 0 hrs
Ex + 4 hrs
Ex + 8 hrs
Pg/mg (Soleus Muscle)
a
800
500 400
*
a
600
a
#
a
#
300
a
#
a
a a b
a
a
a
#
a
# *
a
# *
C T D DT
200 100 0
Ex + 0 hrs
Ex + 4 hrs
aaaa
1
a
0.8
a
a a
a
Ex + 8 hrs
Ex + 12 hrs
a
a
a a
C
0.6
T
0.4
D
0.2
DT
0
Ex + 0 hrs
Ex + 4 hrs
1400
Ex + 8 hrs Ex + 12 hrs
Training:P=0.001 Diabetes: P=0.001 Time Course:P=0.03 Intraction: P=0.02
1600
a
700
1.2
Training:P=0.03 Diabetes: P=0.63 Time Course:P=0.97 Intraction: P=0.92
a
a
a
(D) IL-15 Protein -FHL
Training:P=0.00 Diabetes: P=0.05 Time Course:P=0.01 a Intraction: P=0.81
900
1.4
Ex + 12 hrs
(C) IL-15 Protein - Soleus 1000
(B) IL-15 mRNA - FHL
Pg/mg (FHL Muscle)
Relative Expression IL-15 Level Soleus Muscle
3.5
Relative Expression IL-15 Level FHL Muscle
(A) IL-15 mRNA - Soleus
b
a,b
C T D DT
1200 1000 800 600 400
a,b
a a
* #
a
*
b a
a a,b a a
* * *
* #
Ex + 4 hrs
Ex + 8 hrs
a
*
b a a * *
*
200
0
Ex + 0 hrs
Ex + 12 hrs
Fig. 2 Time course of acute resistance exercise on IL-15 expression in normal and diabetic trained rats. Panels a, b: IL-15 mRNA expression in soleus (a) and flexorhalluces longus (FHL; b); c, d: IL15 protein expression in pg.mg-1 tissue protein. Groups are C healthy control, sedentary rats; T, trained healthy rats; d, diabetic sedentary rats; DT diabetic trained rats. Ex ? 0 h: Immediately after exercise; Ex ? 4 h: 4 h after exercise; Ex ? 8 h: 8 h after exercise, Ex ? 12 h: 12 h after exercise. Results are expressed as
mean ± standard error (SE). Results from the three-way and oneway ANOVAs are presented. For each panel, bars with different superscripts are significantly different (P \ 0.05). a, b bars show differences within different time course groups; hash and asterisk show differences within each time point. Asterisk differ from DT group, hash differ from T group. RNA values were normalized to GAPDH and RPL-26 levels, and expressed as fold change from the C group for each muscle. N = 2–4 animals per group
soleus and FHL muscles increased due to training in both control and diabetic rats. However, post hoc analyses indicated differences in the temporal responses of IL-15 expression in normal and diabetic rats, with biphasic responses observed for diabetic soleus and both control and diabetic FHL muscles. In summary, post-exercise changes in muscle IL-15 mRNA expression and protein levels were discordant, with the general increase in muscle IL-15 protein due to training not reflected by corresponding increases in muscle IL-15 mRNA expression.
expression in the slow soleus muscle (Fig. 3a). IL-6 mRNA expression in the fast FHL muscle exhibited significant effects of training, as well as for diabetes and time course (Fig. 3b). IL-6 mRNA expression for the T group peaked at 4 h after last bout of resistance exercise in the FHL.
IL-6 mRNA expression in FHL and soleus muscles Trained normal and diabetic animals have shown differential IL-6 mRNA expression in slow- and fast-twitch skeletal muscles sampled 24 h following resistance exercise [11]. In this study, we observed a significant effect of training, but not diabetes or time, for IL-15 mRNA
Discussion Our hypothesis was that post-exercise changes in skeletal muscle, IL-15 mRNA expression versus protein levels would differ, as would those of normal and diabetic rats. We observed that IL-15 protein levels increased in both the slow soleus and fast FHL muscles following the last session of a 5-week program of resistance training, although with somewhat different time courses in normal and diabetic rats. However, muscle IL-15 mRNA expression in response to training exhibited a complex pattern that did
123
Endocrine
Training:P=0.002 Diabetes: P=0.1 Time Course:P=0.15 Intraction: P=0.76
Relative Expression IL-6 Level Soleus Muscle
7
a
6
a
5
a
4 3
a
a * a
2
*
1 0
*
Ex + 0 hrs
a a
a a
*
a
*
Ex + 4 hrs
a
a a a
Ex + 8 hrs
a
*
*
C T D DT
Ex + 12 hrs
Training:P=0.03 Diabetes: P=0.007 Time Course:P=0.01 Intraction: P=0.02
(B) IL-6 mRNA - FHL b
4.5
Relative Expression IL-6 Level FHL Muscle
(A) IL-6 mRNA - Soleus
4 3.5 3 2.5
a
a
#
2 1.5 1
a
a a
a
#
#
C
a
a a a aa a
T
a
a
D DT
0.5 0
Ex + 0 hrs
Ex + 4 hrs
Ex + 8 hrs
Ex + 12 hrs
Fig. 3 IL-6 mRNA expression in slow and fast skeletal muscle from the four groups of rats in different time courses after acute resistance exercise in trained normal and diabetic rats. a, b: IL-6 mRNA expression in soleus and FHL muscles; three-way and one-way
ANOVAs revealed significant effects of training for IL-6 mRNA expression in soleus muscle and significant effects of diabetes, training, time course and interaction between training, diabetes, and time course for IL-6 in FHL muscle
not mirror changes in IL-15 protein levels in either muscle type. In contrast, training induced increases in expression of IL-6 mRNA in both muscle types. Other studies have demonstrated a lack of correlation between muscle IL-15 mRNA and protein levels [26, 27], which was confirmed in our study. The lack of correlation of IL-15 mRNA and protein levels has been observed in other tissues and is generally attributed to transcriptional and translational blocks which are presented in the IL-15 gene sequence [7, 26, 27]. Following exercise training, we have observed a complex pattern of muscle IL-15 mRNA response. This behavior is in contrast to the expected upregulation of muscle IL-6 mRNA by exercise training that we observed and that others have documented [28, 29]. In apparent contrast to our findings, other published studies indicated that IL-15 mRNA expression has not changed 24 h after resistance training in [12, 22]. Additionally, one study using human subjects found that IL-15 mRNA content was upregulated in vastus lateralis skeletal muscle 24 h following a single bout of resistance exercise, but was not accompanied by an increase in muscular IL-15 protein level [21]. This same study found no differences in IL-15 mRNA and protein expression at 6 and 48 h after an acute strength exercise [21]. Our finding suggests that the interval between the training session and sampling of the muscle tissue may be a factor in these divergent findings, as we observed significant effects of sampling time on both IL-15 mRNA expression and protein levels in both the soleus and FHL muscles. Therefore, given that the myogenic response to exercise is transient, the optimal timing for muscle biopsy studies is important. Moreover, our findings clearly indicate that muscle IL-15 mRNA expression cannot be used as an index of the effects of different training regimens on intramuscular IL-15 protein levels.
In our study, resistance training in both healthy and diabetic rats increased IL-15 protein levels in the FHL and soleus muscles. Similarly, Kim et al. showed IL-15 protein expression in skeletal muscles, especially in the soleus muscle, significantly increased after treadmill exercise in diabetic animals [13]. Treadmill running is an endurancetype exercise, which would be expected to preferentially affect slow muscles such as the soleus. In the present study, the exercise intervention was progressive ladder climbing, a resistance type of training previously found to increase mass of the fast FHL muscle and not that of other muscle groups [12, 20, 23]. In our study, IL-15 protein levels rose to slightly higher levels in the FHL muscle than in the soleus muscle, but were nevertheless increased in both muscle types. These findings suggest that increases in IL-15 protein expression are only partially related to the type of skeletal muscle that was affected by the exercise training. Training status could also influence myokine mRNA and protein expression after acute exercise. Chronic resistance training involves multiple adaptations including increased muscle mass, improved neuromuscular efficiency, and enhanced muscle metabolism [30]. It has been suggested that the transient changes in transcription during recovery from acute bouts of exercise may accumulate and translate into cellular training adaptations if the exercise is performed for a prolonged period of time [31, 32]. Therefore, changes in IL-15 mRNA expression after resistance exercise training would be predicted; however, while this was observed for IL-6, it was not consistently observed for IL-15. Quinn et al. found that in untrained mice, muscle IL-15 mRNA expression declined following an acute bout of running exercise, while the same stimulus increased circulating IL-15 levels, presumably due to contraction-induced secretion of IL-15 from muscle tissue [33]. Thus, exercise training may function to increase
123
Endocrine
intracellular pools of IL-15 protein to enhance contractioninduced IL-15 release from skeletal muscle. However, one study found no difference in post-exercise increases in serum IL-15 levels between untrained and trained human subjects [34]. Therefore, the functional implications of the increase in muscle IL-15 protein levels in healthy and diabetic animals remain an area for future investigation. The temporal differences in FHL IL-15 protein content between trained healthy and diabetic rats we observed, characterized by prolonged upregulation of IL-15 levels in trained diabetic rats, could be indicative of a block to IL-15 secretion or signaling in diabetic animals. Both acute and chronic exercises are known to elicit muscle damage [35, 36]. We did not assess muscle damage in our study. However, muscle protein content per unit muscle wet weight did not exhibit any differences between trained and untrained groups (not shown), suggesting that edema was not induced. Moreover, in our study, we cannot rule out the possibility that expression of cytokines was derived from non-muscle cells such as infiltrating macrophages and other inflammatory cells, rather than muscle fibers themselves. However, cell culture and in situ hybridization studies have shown that IL-15 and IL-6 are all expressed by muscle fibers at high levels and have been demonstrated to be regulated by exercise and other immune stressors [6, 21, 22, 37]. Evaluation of how much of this effect is due to muscle cells versus inflammatory cells will require further investigation. In this regard, one study demonstrated that the effects of IL-15 on body composition can be independent of lymphocytes [10]. In summary, resistance exercise training inhibited diabetes-induced atrophy of the fast FHL muscle in STZinduced type 1 diabetic rats. Moreover, training, skeletal muscle type, and metabolic status all influence the temporal pattern of post-exercise changes in IL-15 expression. Muscle IL-15 protein levels increase following training, suggesting this may be an adaptation contributing to increased capacity for secretion of this myokine that is not depressed by the diabetic state. Acknowledgments This work was supported by the Research Center of Tarbiat Modares University (TMU), Tehran, Iran. We wish to thank Professor Yaghob Fathoallahy for his help and sincere cooperation. Conflict of interest The authors of this research article have no financial and personal conflict of interest statement.
References 1. B.C. Frier, E.G. Noble, M. Locke, Diabetes-induced atrophy is associated with a muscle-specific alteration in NF-kappaB activation and expression. Cell Stress Chaperones 13(3), 287–296 (2008)
2. H. Andersen, P.C. Gadeberg, B. Brock, J. Jakobsen, Muscular atrophy in diabetic neuropathy: a stereological magnetic resonance imaging study. Diabetologia 40(9), 1062–1069 (1997) 3. T.J. Hawke, D.J. Garry, Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91, 534–551 (2001) 4. P. Mun˜oz-Ca´noves, C. Scheele, B.K. Pedersen, A.L. Serrano, Interleukin-6 myokine signaling in skeletal muscle: a doubleedged sword? FEBS J. 280(17), 4131–4148 (2013) 5. T. Henriksen, C. Green, B.K. Pedersen, Myokines in myogenesis and health. Recent Pat. Biotechnol. 6(3), 167–171 (2012) 6. L.S. Quinn, L. Strait-Bodey, B.G. Anderson, J.M. Argile´s, P.J. Havel, Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway. Cell Biol. Int. 29, 449–457 (2005) 7. T.A. Fehniger, M.A. Caligiuri, Interleukin-15: biology and relevance to human disease. Blood 97(4), 14–32 (2001) 8. N.G. Barra, M.V. Chew, S. Reid, A.A. Ashkar, Interleukin-15 treatment induces weight loss independent of lymphocytes. PLoS ONE 7(6), e39553 (2012) 9. L.S. Quinn, B.G. Anderson, J.D. Conner, E.E. Pistilli, T. WoldenHanson, Overexpression of interleukin-15 in mice promotes resistance to diet-induced obesity, increased insulin sensitivity, and markers of oxidative skeletal muscle metabolism. Int J Interferon, Cytokine Mediat Res 3, 29–42 (2011) 10. N.G. Barra, M.V. Chew, A.C. Holloway, A.A. Ashkar, Interleukin-15 treatment improves glucose homeostasis and insulin sensitivity in obese mice. Diabetes Obes. Metab. 14, 190–193 (2012) 11. S. Busquets, M. Figueras, V. Almendro, F.J. Lopez-Soriano, J.M. Arg-iles, Interleukin-15 increases glucose uptake in skeletal muscle. An antidiabetogenic effect of the cytokine. Biochim. Biophys. Acta 1760, 1613–1617 (2006) 12. M.M. Shamsi, Z.H. Hassan, R. Gharakhanlou, L.S. Quinn, K. Azadmanesh, L. Baghersad et al., Expression of interleukin-15 and inflammatory cytokines in skeletal muscles of STZ-induced diabetic rats: effect of resistance exercise training. Endocrine 46(1), 60–69 (2014) 13. H.J. Kim, J.Y. Park, S.L. Oh, Y.A. Kim, B. So, J.K. Seong, W. Song, Effect of treadmill exercise on interleukin-15 expression and glucose tolerance in zucker diabetic fatty rats. Diabetes Metab J 37(5), 358–364 (2013) 14. B.K. Pedersen, C.P. Fischer, Physiological roles of musclederived interleukin-6 in response to exercise. Curr Opin Clin Nutr Metab Care 10, 265–271 (2007) 15. C. Keller, A. Steensberg, H. Pilegaard, T. Osada, B. Saltin, B.K. Pedersen et al., Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15(14), 2748–2750 (2001) 16. M.A. Febbraio, B.K. Pedersen, Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J 16, 1335–1347 (2002) 17. Q.S. Tao, H.L. Huang, Y. Chai, X. Luo, X.L. Zhang, B. Jia et al., Interleukin-6 up-regulates the expression of interleukin15 is associated with MAPKsand PI3-K signaling pathways in the human keratinocyte cell line, HaCaT. Mol Biol Rep 39(4), 4201–4205 (2012) 18. S.Q. Zhang, X. Luo, S. Yang, J.L. Liu, C.J. Yang, X.Y. Yin et al., Auto inhibition of IL-15 expression in KC cells is ERK1/2 and PI3K dependent. Basic Immun 68, 397–404 (2008) 19. P.A. Farrell, M.J. Fedele, J. Hernandez, J.D. Fluckey, J.L. Miller 3rd, C.H. Lang et al., Hypertrophy of skeletal muscle in diabetic rats in response to chronic resistance exercise. J. Appl. Physiol. 87(3), 1075–1082 (1999) 20. E. Talebi-Garakani, A. Safarzade, Resistance training decreases serum inflammatory markers in diabetic rats. Endocrine 43(3), 564–570 (2013)
123
Endocrine 21. A.R. Nielsen, R. Mounier, P. Plomgaard, O.H. Mortensen, M. Penkowa, T. Speerschneider et al., Expression of interleukin-15 in human skeletal muscle effect of exercise and muscle fibre type composition. J Physiol 584, 305–312 (2007) 22. N.E. Zanchi, F.S. Lira, M.A. de Siqueira Filho, J.C. Rosa, C.R. de Oliveira Carvalho, M. Seelaender et al., Chronic low frequency/ low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle. Eur J Appl Physiol 109(6), 1095–1102 (2010) 23. S. Lee, E.R. Barton, H.L. Sweeney, R.P. Farrar, Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J. Appl. Physiol. 96(3), 1097–1104 (2004) 24. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4), 402–408 (2001) 25. M.M. Bradford, A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976) 26. L.S. Quinn, B.G. Anderson, L. Strait-Bodey, T. Wolden-Hanson, Serum and muscle interleukin-15 levels decrease in aging mice: correlation with declines in soluble interleukin-15 receptor alpha expression. Exp. Gerontol. 45(2), 106–112 (2010) 27. E. Marzetti, C.S. Carter, S.E. Wohlgemuth, H.A. Lees, S. Giovannini, B. Anderson et al., Changes in IL-15 expression and death-receptor apoptotic signaling in rat gastrocnemius muscle with aging and life-long calorie restriction. Mech. Ageing Dev. 130(4), 272–280 (2009) 28. G. Begue, A. Douillard, O. Galbes, B. Rossano, B. Vernus, R. Candau et al., Early activation of rat skeletal muscle IL-6/ STAT1/STAT3 dependent gene expression in resistance exercise linked to hypertrophy. PLoS ONE 8(2), e57141 (2013) 29. E.E. Spangenburg, D.A. Brown, Johnson, R.L. Moore, Exercise increases SOCS-3 expression in rat skeletal muscle: potential relationship to IL-6 expression. J Physiol 572, 839–848 (2006)
123
30. M. Izquierdo, J. Iban˜ez, J.A.L. Calbet, I. Navarro-Amezqueta, M. Gonza´lez-Izal, F. Idoate et al., Cytokine and hormone responses to resistance training. Eur. J. Appl. Physiol. 107, 397–409 (2009) 31. M. Flueck, Tuning of mitochondrial pathways by muscle work: from triggers to sensors and expression signatures. Appl. Physiol. Nutr. Metab. 34, 447–453 (2009) 32. H. Pilegaard, G.A. Ordway, B. Saltin, P.D. Neufer, Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279, E806–E814 (2000) 33. L.S. Quinn, B.G. Anderson, J.D. Conner, T. Wolden-Hanson, T.J. Marcell, IL-15 is required for postexercise induction of the prooxidative mediators PPARd and SIRT1 in male mice. Endocrinology 155(1), 143–155 (2014) 34. S.E. Riechman, G. Balasekaran, S.M. Roth, R.E. Ferrell, Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses. J Appl Physiol 97(6), 2214–2219 (2004) 35. R.S. Staron, R.S. Hikida, T.F. Murray, M.M. Nelson, P. Johnson, F.C. Hagerman, Assessment of skeletal muscle damage in successive biopsies from strength-trained and untrained men and women. Eur. J. Appl. Physiol. 65, 258–264 (1992) 36. S.M. Roth, G.F. Martel, F.M. Ivey, J.T. Lemmer, E.J. Metter, B.F. Hurley, M.A. Rogers, High-volume, heavy-resistance strength training and muscle damage in young and older women. J. Appl. Physiol. 88(3), 1112–1118 (2000) 37. L.S. Quinn, B.G. Anderson, R.H. Drivdahl, B. Alvarez, J.M. Argile´s, Overexpression of interleukin15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders. Exp Cell Res 280(1), 55–63 (2002)
ORIGINAL ARTICLE
Time course of IL-15 expression after acute resistance exercise in trained rats: effect of diabetes and skeletal muscle phenotype Mahdieh Molanouri Shamsi • Zuhair Mohammad Hassan LeBris S. Quinn • Reza Gharakhanlou • Leila Baghersad • Mehdi Mahdavi
•
Received: 14 September 2014 / Accepted: 3 December 2014 Ó Springer Science+Business Media New York 2014
Abstract Type 1 diabetes is associated with skeletal muscle atrophy. Skeletal muscle is an endocrine organ producing myokines such as interleukin-15 (IL-15) and interleukin-6 (IL-6) in response to contraction. These factors may mediate the effects of exercise on skeletal muscle metabolism and anabolic pathways. Lack of correlation between muscle IL-15 mRNA and protein levels after exercise training has been observed, while regulatory effects of IL-6 on IL-15 expression have also been suggested. This study determined post-exercise changes in muscle IL-15 and IL-6 mRNA expression and IL-15 protein levels in healthy and streptozotocin-induced diabetic rats in both the fast flexor hallucis longus (FHL) and slow soleus muscles. Resistance training preserved FHL muscle weight in diabetic rats and increased IL-15 protein levels in both the soleus and FHL muscles. However, the temporal pattern of this response was distinct in normal and diabetic rats. Moreover, discordance between post-exercise muscle IL-15 mRNA and protein expression was observed in
our study, and diabetes suppressed post-exercise increases in FHL muscle IL-6 mRNA expression. Our study indicates that training, skeletal muscle phenotype, and metabolic status all influence the temporal pattern of post-exercise changes in IL15 expression. Muscle IL-15 protein levels increase following training, suggesting this may be an adaptation contributing to increased capacity for secretion of this myokine that is not depressed by the diabetic state.
M. Molanouri Shamsi (&) R. Gharakhanlou L. Baghersad Physical Education & Sport Sciences Department, Faculty of Humanities, Tarbiat Modares University, Tehran, Islamic Republic of Iran e-mail: [email protected]
Z. M. Hassan Jala Ale Ahmad Exp., P.O. Box 14115-13116, Tehran, Islamic Republic of Iran
R. Gharakhanlou e-mail: [email protected] L. Baghersad e-mail: [email protected] M. Molanouri Shamsi R. Gharakhanlou L. Baghersad Jala Ale Ahmad Exp., P.O. Box 14117-13116, Tehran, Islamic Republic of Iran
Keywords Interleukin-15 Interleukin-6 Resistance training Type-1 diabetes Exercise Myokines Cytokines Introduction Skeletal muscle atrophy is considerable in type 1 diabetes [1, 2]. In healthy adults, skeletal muscle displays a remarkable capacity for growth, adaptation, and regeneration in response
L. S. Quinn Division of Gerontology and Geriatric Medicine, Department of Medicine, Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System, University of Washington, Seattle, WA 98108, USA e-mail: [email protected] M. Mahdavi (&) Immunology Department, Pasteur Institute of Iran, 69 Pasteur Ave, Tehran, Iran e-mail: [email protected]
Z. M. Hassan Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Islamic Republic of Iran e-mail: [email protected]
123
Endocrine
to exercise and changes in load [3]. Skeletal muscle produces myokines in response to contraction that may thereby provide a feedback loop for the muscle to regulate its own growth and regeneration, allowing for adaptation to exercise training [4, 5]. Interleukin-15 (IL-15) is a recently discovered myokine that is highly expressed in skeletal muscle tissue [6, 7]. It has been shown that IL-15 can affect body composition independent of immune cells [8]. In addition to its anabolic effects on skeletal muscle, it has been suggested that IL-15 may modulate insulin sensitivity in skeletal muscle [9–11]. Recently, we have shown that diabetes modulates the effects of exercise on IL-15 expression in skeletal muscle [12]. While skeletal muscle IL-15 protein levels increased after chronic exercise training in diabetic rats, comparable changes were not observed for IL-15 mRNA expression, which may be attributed to the complex regulation of IL-15 transcription and translation [7, 12, 13]. IL-6 has also been suggested to function as an exercise-related myokine that has metabolic and hypertrophic actions in skeletal muscle [14]. IL-6 transcription and mRNA expression are markedly upregulated in skeletal muscle during exercise [15], and IL-6 appears to signal through autocrine, paracrine, and hormonal mechanisms to modulate muscle mass and metabolism [5, 16]. Regulatory effects of IL-6 on IL-15 expression have been observed in some studies [17, 18]. Resistance-type exercise training has been shown to represent an effective interventional strategy to augment muscle mass, strength, and function in diabetes [12, 19, 20]. Skeletal muscle tissue is sensitive to the acute and chronic stresses associated with resistance training. These responses are influenced by the structure of resistance activity as well as the training history of the individuals involved. At present, there is conflicting evidence about the exercise-induced changes of IL-15 mRNA and protein in skeletal muscle by training. Some of the disparities reported in the literature may be due to differences in post-exercise muscle sampling intervals, which range from immediate to 24 h post-exercise [12, 21, 22]. In this study, we hypothesized that post-exercise changes in muscle IL-15 mRNA versus IL-15 protein levels would be discordant, and further would be temporally distinct in normal and diabetic rats. Our findings indicate that diabetes, training status, and skeletal muscle type can all modulate post-exercise changes in myokine expression, but that the training-induced increase in muscle IL-15 content is preserved in diabetes.
other scientific purposes, and the protocol was approved by the Ethics Committee of the School of Medicine Sciences, Tarbiat Modares University (TMU), Tehran, Iran. Male Wistar rats weighing 250–280 g were used in this study and were housed in a light- and temperature-controlled animal facility with food and water provided ad lib. Animals were maintained in the Central Animal House, School of Medical Sciences of TMU. Experimental design Animals were randomly assigned to the following treatment groups: control (C, 12 per group); trained (T, 16 per group); STZ-induced diabetes (D, 12 per group); and STZinduced diabetes plus training (DT, 16 per group). Diabetes was induced with IP injection of STZ (Sigma) at 55 mg kg-1 of body weight (BW) in a 0.1 M citrate buffer (pH 4.5). An equal volume of buffer was injected into the control rats. Blood glucose concentrations were assessed after 4 days to ensure that fasting levels greater than 14 mmol l-1 (250 mg dl-1) were reached. Diabetic rats were not treated with insulin during the study, and they showed symptoms of type1 diabetes, such as polyuria and weight loss. Rats in the T and DT groups were trained using a ladder-climbing protocol with progressively larger weight loads attached to the tail, as previously described [23]. Briefly, animal climbed 26 rungs across a 1-m ladder. One repetition along the ladder required 26 total lifts by the animal (13 lifts per limb). Rats were familiarized with the exercise for 3 days, 48 h before STZ injection, and exercise training was initiated after STZ injection. Rats were positioned at the bottom of climbing apparatus and motivated to climb the ladder by touching the tail. Animals were given exercise training with a rest of 48 h between each exercise session. Animals from the T and DT groups were exercised with five sets of four repetitions each with a 60-s rest interval between the reps and 3 min between the sets per session. At 13 and 14 sessions, rats were decreased to 3 sets of 5 repetitions. Weight loads were based on pilot studies for T and DT groups and previously described [12, 20] and are provided in Fig. 1. Based on our pilot studies and previous literature reports [12, 20, 21], it was not possible to use the same loads for healthy and diabetic rats. Tissue preparation
Methods Animals All experiments involving animals were conducted according to the policies of the Iranian convention for the protection of vertebrate animals used for experimental and
123
On the day of sacrifice, rats were randomly divided into four subgroups, C, T, D, and DT groups which, respectively, were sacrificed immediately, 4, 8, and 12 h following the last session of resistance training. Rats were anesthetized with a mixture of Ketamine (30–50 mg kg-1 BW, IP) and Xylazine (3–5 mg kg-1 BW, IP). The soleus and flexor hallucis longus (FHL) muscles, used as
Endocrine 160 140
Percent of Body Mass
expression levels of IL-15 and IL-6 were normalized by subtracting the corresponding levels of the mean of GAPDH and RPL-26 DCT, which were amplified as housekeeping genes. Data are represented as fold change from the C group [24].
Diabetic Nondiabetic
120 100 80
Muscle preparation
60 40 20 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17
Sessions Fig. 1 Protocol for resistance training using ladder climbing. Amount of weight lifted by diabetic and normal rats during 17 sessions of resistance exercise
representative fast-twitch and slow-twitch muscles, respectively, were quickly extracted, weighed, and stored in liquid N2 for subsequent analysis.
Frozen tissues (0.1–0.3 g) were homogenized in RIPA buffer (0.625 % Nonidet P-40, 0.625 % sodium deoxycholate, 6.25 mM sodium phosphate, and 1 mM EDTA at pH 7.4) containing 10 lg ml-1 of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri). Homogenates were centrifuged at 12,000 g for 10 min at 4 °C, the supernatant was saved, and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, California) with bovine serum albumin as a Ref. [25]. Supernatants were aliquotted and stored at -80 °C. Assay of IL-15 protein in skeletal muscles
Real-time PCR Total RNA was extracted from frozen soleus and FHL muscles samples using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s recommendations. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity was determined by calculating the absorbance ratio at 260 and 280 nm and by ethidium bromide staining. Purification was accepted when the 260/280 nm absorbance ratio was above 1.8. Isolated RNA was stored at -80 °C. RNA was reverse transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent Kit (Perfect Real Time, Takara Code RR037A, Japan) using the following protocol: reverse transcription at 37 °C for 15 min, inactivation of reverse transcriptase at 85 °C for 5 s, and refrigeration at 4 °C for 10 min, with storage at -20 °C. Primer sets for rat GAPDH (glyceraldehyde 3-phosphate dehydrogenase), RPL-26 (ribosomal protein L26), IL-15, and IL-6 were produced by Qiagen QuantiTect (GAPDH, QT00199633; RPL-26, QT01828771; IL-15, QT01813637 and IL-6, QT00182896). Gene expression was measured by real-time PCR using the Rotor-Gene 6000 (Corbett Research, Mortlake, Australia). The thermal cycle protocol was as follows: 1 cycle at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 30 s. PCR amplification was performed in duplicate in a total reaction volume of 20 ll. The reaction mixture consisted of 3 ll diluted template, 10 ll SYBR Premix Ex TaqTMKit (Perfect Real Time, Takara Code RR041A, Japan), and 2 ll primers. Amplification specificity was controlled by a melting curve analysis and gel electrophoresis of the PCR products. Relative
IL-15 protein concentrations in muscle were determined in duplicate using the USCN ELISA kit (USCN Life Science Inc., Wuhan,China or Houston, TX). The assays were carried out according to the manufacturers’ instructions. Muscle IL-15 data are expressed as pg of cytokine per mg of total protein. The minimum detectable concentration was \5.6 pg ml-1 for IL-15. The intra- and inter-assay coefficients of variation were 10 and 12 % for IL-15, respectively. Blood glucose and serum insulin measurements Fasting blood was sampled from the tail vein in rats before sacrifice. Blood glucose levels were tested by glucometer (GT-1920, Japan) with samples run in duplicate. Serum insulin concentrations were measured using a commercially available ultrasensitive rat insulin ELISA kit (ALPCO Diagnostics, Windham, NH). Statistical analysis All analyses were performed using SPSS V16.0 (SPSS, Chicago, IL). Three-way analysis of variance (three-way ANOVA) and Tukey post hoc tests were used for phenotypic, mRNA, and protein data in different time course following resistance exercise. Also, one-way ANOVA and Tukey post doc tests were used for mRNA and protein data in different groups per time course following resistance exercise. Statistical significance was set at P \ 0.05. Data are presented as mean ± SEM.
123
123
50.8 ± 2.3a
Within rows, values with different letters are different at P \ 0.05; values with the same letter are not significantly different
48.4 ± 1.8a 49.3 ± 2.4a 51.7 ± 2.6a 47.8 ± 1.5a
IW initial weight (g), FW final weight (g), FG fasting glucose (mmol l ), FI fasting insulin (ng ml ), FMM flexor hallucis longus (FHL), muscle mass (mg), FMBR FHL-to-body mass ratio (mg g-1 9 100), SMM soleus muscle mass (mg), SMBR soleus-to-body mass ratio (mg g-1 9 100)
-1
50.06 ± 3.5a 52.1 ± 1.7a 46.6 ± 3.7a SMBR
47.8 ± 2.8a
664.5 ± 7.8b
4.5 ± 0.24a
211.5 ± 6.5b 159.7 ± 5.7a 212.1 ± 3.5b 155.2 ± 8.7a
659.7 ± 12.3b 663.5 ± 15.3b
213.3 ± 5.4b 163.2 ± 11.4a 207.4 ± 7.6b 157.2 ± 16.9a 173.2 ± 3.7a 160.4 ± 14.3a FMBR SMM
676.2 ± 39.3b 556.6 ± 22.6a FMM
0.65 ± 0.05a
4.4 ± 0.1a 4.7 ± 0.1a
0.78 ± 0.06a 0.7 ± 0.07a
4.6 ± 0.1a
0.6 ± 0.04a
FG
FI
4.7 ± 0.04a
0.6 ± 0.03a
-1
46.06 ± 1.1a
199.4 ± 7.3b 118.7 ± 7.1b
481.5 ± 8.6d 477.5 ± 8.2d
207.3 ± 8.8b 114.5 ± 9.2b 202 ± 7.3b 121.7 ± 7.8b
471 ± 10.7d 474 ± 4.3d
201.1 ± 5.2b 113 ± 4.6b 168.2 ± 7.4a 110.4 ± 3.02b
400.8 ± 11.8c
27.1 ± 1.5b
0.16 ± 0.02b 0.18 ± 0.05b
26.8 ± 1.6b 28.2 ± 2.8b
0.21 ± 0.02b 0.19 ± 0.04b
27.2 ± 2.1b 30.1 ± 1.1b
261 ± 9.2a 266 ± 7.1a
232 ± 13.3b 235.7 ± 13.7b
262.5 ± 6.2a 267.5 ± 10.5a
236.2 ± 8. 6b 315.7 ± 12.9a
263.7 ± 8a 265.7 ± 8a
311.5 ± 10.8a 311.7 ± 11.8a
265.5 ± 8a 269 ± 9.4a
328.2 ± 27.7a
267.1 ± 5.3a
320.3 ± 7.5a
IW
FW
T12 T4 T0 C
Table 1 Characteristic features of rats in different groups
T8
IL-15 mRNA and protein expression in FHL and soleus muscles To determine the effects of training, diabetes, and time course on IL-15 mRNA and protein expression in slowand fast-twitch skeletal muscles, diabetic and normal rats were trained for 5 weeks with ladder climbing and sacrificed at different time points up to 12 h following the last session of resistance training. In the soleus muscle, a significant effect of diabetes, but not training or time, on IL15 mRNA expression was observed (Fig. 2a). Furthermore, we did not observe significant time-dependent differences within each of the four treatment groups for the soleus muscle (P [ 0.05), while immediately following exercise, IL-15 mRNA expression was significantly decreased in the DT group compared to controls (Fig. 2a). IL-15 mRNA expression in the fast FHL muscle exhibited a significant effect of diabetes, training, and time course, with the training-induced increase in IL-15 mRNA expression at 4 h blunted in the DT group (Fig. 2b). In contrast to the results for IL-15 mRNA expression, IL-15 protein levels in both FHL and soleus muscle showed significant effects of diabetes, training, and time. (Figure 2 c, d). Overall, IL-15 protein levels in both the
0.17 ± 0.03b
D
DT0
DT4
DT8
Rats underwent the protocols for controls (C0, C4, C8, and C12), resistance training (T0, T4, T8, and T12), STZinduced diabetes (D0, D4, D8, and D12) and diabetes with resistance training (DT0, DT4, DT8, and DT12) as outlined in Methods. The characteristic features of the rats in different groups for the first study are presented in Table 1, which shows that the diabetic groups displayed significant reductions in plasma insulin levels compared to the healthy groups. Consequently, typical type 1 diabetes hyperglycemia occurred in the diabetic groups, with the increase in fasting glucose. Moreover, at the end of the study, diabetic rats showed a significant decrease in total BW, although different groups did not have noticeable differences in total weight at the beginning of the study (Table 1). Mass of both the soleus and FHL muscles was lower in diabetic animals compared to controls (Table 1). Five weeks of resistance training resulted in hypertrophy of the fast FHL muscle in both normal and diabetic training groups, as indicated by a significantly higher FHL mass and FHL mass to body mass ratio (FMBR) in the trained animals compared to the sedentary animals (Table 1). Soleus muscles did not undergo hypertrophy in response to resistance training (Table 1).
263.2 ± 5.5a
DT12
Induction of diabetes mellitus and effects of training on muscle mass
241.4 ± 1b
Results
242.7 ± 12.7b
Endocrine
Endocrine
a
a
3
a
2.5
a
a
2 1.5
Training:P=0.93 Diabetes: P=0.001 Time Course:P=0.1 Intraction: P=0.46
a
*
1
a a
a
a
*
a
a
a
a
C
a a
T D DT
0.5 0
Ex + 0 hrs
Ex + 4 hrs
Ex + 8 hrs
Pg/mg (Soleus Muscle)
a
800
500 400
*
a
600
a
#
a
#
300
a
#
a
a a b
a
a
a
#
a
# *
a
# *
C T D DT
200 100 0
Ex + 0 hrs
Ex + 4 hrs
aaaa
1
a
0.8
a
a a
a
Ex + 8 hrs
Ex + 12 hrs
a
a
a a
C
0.6
T
0.4
D
0.2
DT
0
Ex + 0 hrs
Ex + 4 hrs
1400
Ex + 8 hrs Ex + 12 hrs
Training:P=0.001 Diabetes: P=0.001 Time Course:P=0.03 Intraction: P=0.02
1600
a
700
1.2
Training:P=0.03 Diabetes: P=0.63 Time Course:P=0.97 Intraction: P=0.92
a
a
a
(D) IL-15 Protein -FHL
Training:P=0.00 Diabetes: P=0.05 Time Course:P=0.01 a Intraction: P=0.81
900
1.4
Ex + 12 hrs
(C) IL-15 Protein - Soleus 1000
(B) IL-15 mRNA - FHL
Pg/mg (FHL Muscle)
Relative Expression IL-15 Level Soleus Muscle
3.5
Relative Expression IL-15 Level FHL Muscle
(A) IL-15 mRNA - Soleus
b
a,b
C T D DT
1200 1000 800 600 400
a,b
a a
* #
a
*
b a
a a,b a a
* * *
* #
Ex + 4 hrs
Ex + 8 hrs
a
*
b a a * *
*
200
0
Ex + 0 hrs
Ex + 12 hrs
Fig. 2 Time course of acute resistance exercise on IL-15 expression in normal and diabetic trained rats. Panels a, b: IL-15 mRNA expression in soleus (a) and flexorhalluces longus (FHL; b); c, d: IL15 protein expression in pg.mg-1 tissue protein. Groups are C healthy control, sedentary rats; T, trained healthy rats; d, diabetic sedentary rats; DT diabetic trained rats. Ex ? 0 h: Immediately after exercise; Ex ? 4 h: 4 h after exercise; Ex ? 8 h: 8 h after exercise, Ex ? 12 h: 12 h after exercise. Results are expressed as
mean ± standard error (SE). Results from the three-way and oneway ANOVAs are presented. For each panel, bars with different superscripts are significantly different (P \ 0.05). a, b bars show differences within different time course groups; hash and asterisk show differences within each time point. Asterisk differ from DT group, hash differ from T group. RNA values were normalized to GAPDH and RPL-26 levels, and expressed as fold change from the C group for each muscle. N = 2–4 animals per group
soleus and FHL muscles increased due to training in both control and diabetic rats. However, post hoc analyses indicated differences in the temporal responses of IL-15 expression in normal and diabetic rats, with biphasic responses observed for diabetic soleus and both control and diabetic FHL muscles. In summary, post-exercise changes in muscle IL-15 mRNA expression and protein levels were discordant, with the general increase in muscle IL-15 protein due to training not reflected by corresponding increases in muscle IL-15 mRNA expression.
expression in the slow soleus muscle (Fig. 3a). IL-6 mRNA expression in the fast FHL muscle exhibited significant effects of training, as well as for diabetes and time course (Fig. 3b). IL-6 mRNA expression for the T group peaked at 4 h after last bout of resistance exercise in the FHL.
IL-6 mRNA expression in FHL and soleus muscles Trained normal and diabetic animals have shown differential IL-6 mRNA expression in slow- and fast-twitch skeletal muscles sampled 24 h following resistance exercise [11]. In this study, we observed a significant effect of training, but not diabetes or time, for IL-15 mRNA
Discussion Our hypothesis was that post-exercise changes in skeletal muscle, IL-15 mRNA expression versus protein levels would differ, as would those of normal and diabetic rats. We observed that IL-15 protein levels increased in both the slow soleus and fast FHL muscles following the last session of a 5-week program of resistance training, although with somewhat different time courses in normal and diabetic rats. However, muscle IL-15 mRNA expression in response to training exhibited a complex pattern that did
123
Endocrine
Training:P=0.002 Diabetes: P=0.1 Time Course:P=0.15 Intraction: P=0.76
Relative Expression IL-6 Level Soleus Muscle
7
a
6
a
5
a
4 3
a
a * a
2
*
1 0
*
Ex + 0 hrs
a a
a a
*
a
*
Ex + 4 hrs
a
a a a
Ex + 8 hrs
a
*
*
C T D DT
Ex + 12 hrs
Training:P=0.03 Diabetes: P=0.007 Time Course:P=0.01 Intraction: P=0.02
(B) IL-6 mRNA - FHL b
4.5
Relative Expression IL-6 Level FHL Muscle
(A) IL-6 mRNA - Soleus
4 3.5 3 2.5
a
a
#
2 1.5 1
a
a a
a
#
#
C
a
a a a aa a
T
a
a
D DT
0.5 0
Ex + 0 hrs
Ex + 4 hrs
Ex + 8 hrs
Ex + 12 hrs
Fig. 3 IL-6 mRNA expression in slow and fast skeletal muscle from the four groups of rats in different time courses after acute resistance exercise in trained normal and diabetic rats. a, b: IL-6 mRNA expression in soleus and FHL muscles; three-way and one-way
ANOVAs revealed significant effects of training for IL-6 mRNA expression in soleus muscle and significant effects of diabetes, training, time course and interaction between training, diabetes, and time course for IL-6 in FHL muscle
not mirror changes in IL-15 protein levels in either muscle type. In contrast, training induced increases in expression of IL-6 mRNA in both muscle types. Other studies have demonstrated a lack of correlation between muscle IL-15 mRNA and protein levels [26, 27], which was confirmed in our study. The lack of correlation of IL-15 mRNA and protein levels has been observed in other tissues and is generally attributed to transcriptional and translational blocks which are presented in the IL-15 gene sequence [7, 26, 27]. Following exercise training, we have observed a complex pattern of muscle IL-15 mRNA response. This behavior is in contrast to the expected upregulation of muscle IL-6 mRNA by exercise training that we observed and that others have documented [28, 29]. In apparent contrast to our findings, other published studies indicated that IL-15 mRNA expression has not changed 24 h after resistance training in [12, 22]. Additionally, one study using human subjects found that IL-15 mRNA content was upregulated in vastus lateralis skeletal muscle 24 h following a single bout of resistance exercise, but was not accompanied by an increase in muscular IL-15 protein level [21]. This same study found no differences in IL-15 mRNA and protein expression at 6 and 48 h after an acute strength exercise [21]. Our finding suggests that the interval between the training session and sampling of the muscle tissue may be a factor in these divergent findings, as we observed significant effects of sampling time on both IL-15 mRNA expression and protein levels in both the soleus and FHL muscles. Therefore, given that the myogenic response to exercise is transient, the optimal timing for muscle biopsy studies is important. Moreover, our findings clearly indicate that muscle IL-15 mRNA expression cannot be used as an index of the effects of different training regimens on intramuscular IL-15 protein levels.
In our study, resistance training in both healthy and diabetic rats increased IL-15 protein levels in the FHL and soleus muscles. Similarly, Kim et al. showed IL-15 protein expression in skeletal muscles, especially in the soleus muscle, significantly increased after treadmill exercise in diabetic animals [13]. Treadmill running is an endurancetype exercise, which would be expected to preferentially affect slow muscles such as the soleus. In the present study, the exercise intervention was progressive ladder climbing, a resistance type of training previously found to increase mass of the fast FHL muscle and not that of other muscle groups [12, 20, 23]. In our study, IL-15 protein levels rose to slightly higher levels in the FHL muscle than in the soleus muscle, but were nevertheless increased in both muscle types. These findings suggest that increases in IL-15 protein expression are only partially related to the type of skeletal muscle that was affected by the exercise training. Training status could also influence myokine mRNA and protein expression after acute exercise. Chronic resistance training involves multiple adaptations including increased muscle mass, improved neuromuscular efficiency, and enhanced muscle metabolism [30]. It has been suggested that the transient changes in transcription during recovery from acute bouts of exercise may accumulate and translate into cellular training adaptations if the exercise is performed for a prolonged period of time [31, 32]. Therefore, changes in IL-15 mRNA expression after resistance exercise training would be predicted; however, while this was observed for IL-6, it was not consistently observed for IL-15. Quinn et al. found that in untrained mice, muscle IL-15 mRNA expression declined following an acute bout of running exercise, while the same stimulus increased circulating IL-15 levels, presumably due to contraction-induced secretion of IL-15 from muscle tissue [33]. Thus, exercise training may function to increase
123
Endocrine
intracellular pools of IL-15 protein to enhance contractioninduced IL-15 release from skeletal muscle. However, one study found no difference in post-exercise increases in serum IL-15 levels between untrained and trained human subjects [34]. Therefore, the functional implications of the increase in muscle IL-15 protein levels in healthy and diabetic animals remain an area for future investigation. The temporal differences in FHL IL-15 protein content between trained healthy and diabetic rats we observed, characterized by prolonged upregulation of IL-15 levels in trained diabetic rats, could be indicative of a block to IL-15 secretion or signaling in diabetic animals. Both acute and chronic exercises are known to elicit muscle damage [35, 36]. We did not assess muscle damage in our study. However, muscle protein content per unit muscle wet weight did not exhibit any differences between trained and untrained groups (not shown), suggesting that edema was not induced. Moreover, in our study, we cannot rule out the possibility that expression of cytokines was derived from non-muscle cells such as infiltrating macrophages and other inflammatory cells, rather than muscle fibers themselves. However, cell culture and in situ hybridization studies have shown that IL-15 and IL-6 are all expressed by muscle fibers at high levels and have been demonstrated to be regulated by exercise and other immune stressors [6, 21, 22, 37]. Evaluation of how much of this effect is due to muscle cells versus inflammatory cells will require further investigation. In this regard, one study demonstrated that the effects of IL-15 on body composition can be independent of lymphocytes [10]. In summary, resistance exercise training inhibited diabetes-induced atrophy of the fast FHL muscle in STZinduced type 1 diabetic rats. Moreover, training, skeletal muscle type, and metabolic status all influence the temporal pattern of post-exercise changes in IL-15 expression. Muscle IL-15 protein levels increase following training, suggesting this may be an adaptation contributing to increased capacity for secretion of this myokine that is not depressed by the diabetic state. Acknowledgments This work was supported by the Research Center of Tarbiat Modares University (TMU), Tehran, Iran. We wish to thank Professor Yaghob Fathoallahy for his help and sincere cooperation. Conflict of interest The authors of this research article have no financial and personal conflict of interest statement.
References 1. B.C. Frier, E.G. Noble, M. Locke, Diabetes-induced atrophy is associated with a muscle-specific alteration in NF-kappaB activation and expression. Cell Stress Chaperones 13(3), 287–296 (2008)
2. H. Andersen, P.C. Gadeberg, B. Brock, J. Jakobsen, Muscular atrophy in diabetic neuropathy: a stereological magnetic resonance imaging study. Diabetologia 40(9), 1062–1069 (1997) 3. T.J. Hawke, D.J. Garry, Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91, 534–551 (2001) 4. P. Mun˜oz-Ca´noves, C. Scheele, B.K. Pedersen, A.L. Serrano, Interleukin-6 myokine signaling in skeletal muscle: a doubleedged sword? FEBS J. 280(17), 4131–4148 (2013) 5. T. Henriksen, C. Green, B.K. Pedersen, Myokines in myogenesis and health. Recent Pat. Biotechnol. 6(3), 167–171 (2012) 6. L.S. Quinn, L. Strait-Bodey, B.G. Anderson, J.M. Argile´s, P.J. Havel, Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway. Cell Biol. Int. 29, 449–457 (2005) 7. T.A. Fehniger, M.A. Caligiuri, Interleukin-15: biology and relevance to human disease. Blood 97(4), 14–32 (2001) 8. N.G. Barra, M.V. Chew, S. Reid, A.A. Ashkar, Interleukin-15 treatment induces weight loss independent of lymphocytes. PLoS ONE 7(6), e39553 (2012) 9. L.S. Quinn, B.G. Anderson, J.D. Conner, E.E. Pistilli, T. WoldenHanson, Overexpression of interleukin-15 in mice promotes resistance to diet-induced obesity, increased insulin sensitivity, and markers of oxidative skeletal muscle metabolism. Int J Interferon, Cytokine Mediat Res 3, 29–42 (2011) 10. N.G. Barra, M.V. Chew, A.C. Holloway, A.A. Ashkar, Interleukin-15 treatment improves glucose homeostasis and insulin sensitivity in obese mice. Diabetes Obes. Metab. 14, 190–193 (2012) 11. S. Busquets, M. Figueras, V. Almendro, F.J. Lopez-Soriano, J.M. Arg-iles, Interleukin-15 increases glucose uptake in skeletal muscle. An antidiabetogenic effect of the cytokine. Biochim. Biophys. Acta 1760, 1613–1617 (2006) 12. M.M. Shamsi, Z.H. Hassan, R. Gharakhanlou, L.S. Quinn, K. Azadmanesh, L. Baghersad et al., Expression of interleukin-15 and inflammatory cytokines in skeletal muscles of STZ-induced diabetic rats: effect of resistance exercise training. Endocrine 46(1), 60–69 (2014) 13. H.J. Kim, J.Y. Park, S.L. Oh, Y.A. Kim, B. So, J.K. Seong, W. Song, Effect of treadmill exercise on interleukin-15 expression and glucose tolerance in zucker diabetic fatty rats. Diabetes Metab J 37(5), 358–364 (2013) 14. B.K. Pedersen, C.P. Fischer, Physiological roles of musclederived interleukin-6 in response to exercise. Curr Opin Clin Nutr Metab Care 10, 265–271 (2007) 15. C. Keller, A. Steensberg, H. Pilegaard, T. Osada, B. Saltin, B.K. Pedersen et al., Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J 15(14), 2748–2750 (2001) 16. M.A. Febbraio, B.K. Pedersen, Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J 16, 1335–1347 (2002) 17. Q.S. Tao, H.L. Huang, Y. Chai, X. Luo, X.L. Zhang, B. Jia et al., Interleukin-6 up-regulates the expression of interleukin15 is associated with MAPKsand PI3-K signaling pathways in the human keratinocyte cell line, HaCaT. Mol Biol Rep 39(4), 4201–4205 (2012) 18. S.Q. Zhang, X. Luo, S. Yang, J.L. Liu, C.J. Yang, X.Y. Yin et al., Auto inhibition of IL-15 expression in KC cells is ERK1/2 and PI3K dependent. Basic Immun 68, 397–404 (2008) 19. P.A. Farrell, M.J. Fedele, J. Hernandez, J.D. Fluckey, J.L. Miller 3rd, C.H. Lang et al., Hypertrophy of skeletal muscle in diabetic rats in response to chronic resistance exercise. J. Appl. Physiol. 87(3), 1075–1082 (1999) 20. E. Talebi-Garakani, A. Safarzade, Resistance training decreases serum inflammatory markers in diabetic rats. Endocrine 43(3), 564–570 (2013)
123
Endocrine 21. A.R. Nielsen, R. Mounier, P. Plomgaard, O.H. Mortensen, M. Penkowa, T. Speerschneider et al., Expression of interleukin-15 in human skeletal muscle effect of exercise and muscle fibre type composition. J Physiol 584, 305–312 (2007) 22. N.E. Zanchi, F.S. Lira, M.A. de Siqueira Filho, J.C. Rosa, C.R. de Oliveira Carvalho, M. Seelaender et al., Chronic low frequency/ low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle. Eur J Appl Physiol 109(6), 1095–1102 (2010) 23. S. Lee, E.R. Barton, H.L. Sweeney, R.P. Farrar, Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J. Appl. Physiol. 96(3), 1097–1104 (2004) 24. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4), 402–408 (2001) 25. M.M. Bradford, A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976) 26. L.S. Quinn, B.G. Anderson, L. Strait-Bodey, T. Wolden-Hanson, Serum and muscle interleukin-15 levels decrease in aging mice: correlation with declines in soluble interleukin-15 receptor alpha expression. Exp. Gerontol. 45(2), 106–112 (2010) 27. E. Marzetti, C.S. Carter, S.E. Wohlgemuth, H.A. Lees, S. Giovannini, B. Anderson et al., Changes in IL-15 expression and death-receptor apoptotic signaling in rat gastrocnemius muscle with aging and life-long calorie restriction. Mech. Ageing Dev. 130(4), 272–280 (2009) 28. G. Begue, A. Douillard, O. Galbes, B. Rossano, B. Vernus, R. Candau et al., Early activation of rat skeletal muscle IL-6/ STAT1/STAT3 dependent gene expression in resistance exercise linked to hypertrophy. PLoS ONE 8(2), e57141 (2013) 29. E.E. Spangenburg, D.A. Brown, Johnson, R.L. Moore, Exercise increases SOCS-3 expression in rat skeletal muscle: potential relationship to IL-6 expression. J Physiol 572, 839–848 (2006)
123
30. M. Izquierdo, J. Iban˜ez, J.A.L. Calbet, I. Navarro-Amezqueta, M. Gonza´lez-Izal, F. Idoate et al., Cytokine and hormone responses to resistance training. Eur. J. Appl. Physiol. 107, 397–409 (2009) 31. M. Flueck, Tuning of mitochondrial pathways by muscle work: from triggers to sensors and expression signatures. Appl. Physiol. Nutr. Metab. 34, 447–453 (2009) 32. H. Pilegaard, G.A. Ordway, B. Saltin, P.D. Neufer, Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279, E806–E814 (2000) 33. L.S. Quinn, B.G. Anderson, J.D. Conner, T. Wolden-Hanson, T.J. Marcell, IL-15 is required for postexercise induction of the prooxidative mediators PPARd and SIRT1 in male mice. Endocrinology 155(1), 143–155 (2014) 34. S.E. Riechman, G. Balasekaran, S.M. Roth, R.E. Ferrell, Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses. J Appl Physiol 97(6), 2214–2219 (2004) 35. R.S. Staron, R.S. Hikida, T.F. Murray, M.M. Nelson, P. Johnson, F.C. Hagerman, Assessment of skeletal muscle damage in successive biopsies from strength-trained and untrained men and women. Eur. J. Appl. Physiol. 65, 258–264 (1992) 36. S.M. Roth, G.F. Martel, F.M. Ivey, J.T. Lemmer, E.J. Metter, B.F. Hurley, M.A. Rogers, High-volume, heavy-resistance strength training and muscle damage in young and older women. J. Appl. Physiol. 88(3), 1112–1118 (2000) 37. L.S. Quinn, B.G. Anderson, R.H. Drivdahl, B. Alvarez, J.M. Argile´s, Overexpression of interleukin15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders. Exp Cell Res 280(1), 55–63 (2002)
