J Appl Physiol 97: 2214–2219, 2004; doi:10.1152/japplphysiol.00491.2004.

Association of interleukin-15 protein and interleukin-15 receptor genetic variation with resistance exercise training responses

Steven E. Riechman,1 G. Balasekaran,1 Stephen M. Roth,2 and Robert E. Ferrell1 1Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260; and 2Department of Kinesiology, University of Maryland, College Park, Maryland 20742 Submitted 6 May 2004; accepted in final form 16 July 2004

Riechman, Steven E., G. Balasekaran, Stephen M. Roth, and IL-15 signals through the IL-15 receptor-␣ and is found in a Robert E. Ferrell. Association of interleukin-15 protein and inter- wide variety of tissues including skeletal muscle. In fact, leukin-15 receptor genetic variation with resistance exercise training skeletal muscle levels of IL-15 are among the highest of any responses. J Appl Physiol 97: 2214–2219, 2004; doi:10.1152/ tissue (11, 17), and IL-15 is one of the most abundant cytokines japplphysiol.00491.2004.—Interleukin-15 (IL-15) is an anabolic cy- in skeletal muscle (15). tokine that is produced in skeletal muscle and directly affects muscle IL-15 has been shown to be anabolic for differentiated anabolism in animal and in vitro models. The contribution of IL-15 mouse C2 and cultured bovine primary muscle cells marked by variability in muscle responses to 10 wk of resistance exercise training an increase myosin heavy chain accumulation (18). In a rat in young men and women was examined by measuring acute and chronic changes in IL-15 protein in plasma and characterizing genetic model of cancer cachexia, IL-15 treatment inhibited skeletal variation in the IL-15 receptor-␣ gene (IL15RA). Participants trained muscle wasting by decreasing protein degradation rates 3 days a week at 75% of one repetition maximum, performing three through inhibition of the ATP-ubiquitin-dependent proteolytic sets (6–10 repetitions) of 13 resistance exercises. Plasma IL-15 pathway (6). Similarly, transfection of C2 cells with an IL-15 protein was significantly increased (P Ͻ 0.05) immediately after acute expression vector induced myotube hypertrophy, independent resistance exercise but did not change with training and was not of IGF action, through increases in protein synthesis and associated with variability in muscle responses with training. A single decreases in protein degradation (16). The anabolic properties nucleotide polymorphism in exon 7 of IL15RA was strongly associ- in these models did not include any effect of IL-15 on prolif- ated with muscle hypertrophy and accounted for 7.1% of the variation eration or differentiation of myocytes, although inhibition of in regression modeling. A polymorphism in exon 4 was also indepen- IGF, through a stable transfection of IGF binding protein, dently associated with muscle hypertrophy and accounted for an resulted in elevated differentiation with IL-15 administration to additional 3.5% of the variation in hypertrophy. These results suggest C2 cell cultures (17). that IL-15 is an important mediator of muscle mass response to The anabolic effects of IL-15 on skeletal muscle suggest a resistance exercise training in humans and that genetic variation in promising treatment for muscle loss conditions such as ca- IL15RA accounts for a significant proportion of the variability in this chexia, sarcopenia, and disuse atrophy. However, existing response. studies have been limited to animal and in vitro models, and no IL-15; strength; skeletal muscle; muscle quality study has addressed the role of IL-15 signaling on variability in muscle phenotypes in humans. Therefore, a primary purpose of this investigation was to determine whether genetic variation in RESISTANCE EXERCISE TRAINING (RET) is well known to result in the IL-15 receptor-␣ gene or plasma IL-15 concentrations were marked increases in muscle mass and strength, but the re- associated with the variability in muscle mass, strength, and sponses to a standardized program are considerably variable muscle quality changes after standardized high-intensity RET. among individuals (19). Our understanding of the characteris- tics that account for this interindividual variability in muscle MATERIALS AND METHODS responses is limited. The Molecular Epidemiology of Resis- Subjects. tance Exercise Training (MERET) study was designed to One hundred fifty-three participants completed 10 wk of RET (76 men and 77 women). Eligibility was based on the examine environmental and genetic contributions to these following criteria: age between 18 and 31 yr; no medical history variable muscle responses in young men and women. The for disorders involving the cardiovascular, endocrine, or skeletal present report examined the association of genetic variation in muscle systems; Ͻ3 h of RET activity per week in the past year; the IL-15 receptor-␣ to muscle responses to 10 wk of high- normal caloric consumption; and no report of taking anabolic intensity resistance training as well as the plasma changes in steroids or other putative performance-enhancing supplements. IL-15 protein in response to acute and chronic resistance Participants were instructed not to perform RET activity outside of exercise. the program and to maintain all other non-RET activities at the same level. After all procedures were explained, subjects gave IL-15 was initially identified as a T-cell growth factor and written, informed consent before participation in the study under shares many properties and functions with IL-2 (10), including protocols approved by the University of Pittsburgh Institutional sharing two of three components of their heterotrimeric recep- Review Board. A more detailed description of the cohort and the tor complexes (IL-2 R␤ and IL-2 R␥c). Unlike IL-2, however, intervention has been reported (19).

Address for reprint requests and other correspondence: S. E. Riechman, The costs of publication of this article were defrayed in part by the payment A300 Crabtree Hall, University of Pittsburgh, Pittsburgh, PA 15260 (E-mail: of page charges. The article must therefore be hereby marked “advertisement” [email protected]). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2214 8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society http://www.jap.org IL15RA POLYMORPHISM AND RESISTANCE TRAINING 2215 One repetition maximum. Three familiarization-training sessions For the exon 7 PstI variation, MERET samples were amplified by were conducted before the initiation of the program, using the mini- using forward (ctt ccg tcc ctc atc cta aCc t) and reverse (cca ggt ccc mum weight to induce resistance. After these familiarization sessions, tgt cca tgt gtg) primers. Capital C indicates the mismatch. Each one repetition maximums (1 RMs) were determined after a 3-min 35-cycle PCR reaction was performed using 54°C annealing temper- warm-up on an aerobic exercise machine and stretching. Before a 1 ature and 50-␮l final reaction volumes containing 100–200 ng DNA, RM attempt, subjects performed three warm-up repetitions at a weight 0.46 ␮M of deoxyribonucleotide triphosphate (dNTP) mixture, 50 equivalent to 50% of an estimated 1 RM. The weight was then mM KCl, 10 MM Tris⅐HCl (pH ϭ 9.0), 1.5 mM MgCl, 0.1% Triton increased to 90% of a reestimated 1 RM to perform one repetition. X-100, and 0.8 U of Taq DNA polymerase. After 60 s of rest, the weight was increased to 100% fora1RM Amplified product (16 ␮l) from study subjects was added to5Uof attempt. Thereafter, successful attempts were followed by attempts PstI for digestion overnight at 37°C. Each digested sample was loaded made with a higher resistance in a manner that minimized the total onto a 2.5% agarose gel containing ethidium bromide and electropho- number of attempts required before the 1 RM value was obtained. resed for 3.5 h at 100 V. Internal controls were run on each of these This procedure was performed on all exercise equipment used for the gels to ensure complete digestion by the PstI enzyme. After electro- RET program in the same order for all participants within 4 days of phoresis, the DNA fragments were visualized by ultraviolet illumina- starting and 4 days of completing the RET. tion (Eagle Eye), and fragment sizes were estimated by comparison to Body composition. Baseline and post-RET assessments were con- a 1-kb ladder run on the same gel. The presence of a polymorphic ducted for height (wall-mounted stadiometer), weight (Detecto Clin- restriction site (Pst I) at the IL15RA gene locus is specified as C base, ical scale), body composition by hydrostatic weighing (21), and whereas absence of this site is an A base. Subjects were categorized circumference measurements of arms and thighs. All measurements as exhibiting the CC, CA, or AA genotype. were made with subjects in the minimal attire used to enter the The same methods were used for the exon 4 BstNI and intron 5 underwater weighing tank within 2–4 days of starting or completing HpaII sites with the following exceptions. The fragments containing the RET. Arm circumference was measured at the middle of the bicep the BstNI and HpaII sites were amplified with 50°C annealing with the arm relaxed at the side of the body. Thigh circumference was temperature and the primers F: taa agc tgc ctt gtg ccg gat c R: gct ggt measured at the midpoint of the femur. Duplicate measures were made cag ggt gac tca cta t and F: atc tga tgg agg cct tct gag R: gct tca tga of each circumference and recorded to the nearest 0.1 cm. gga act agg acc, respectively. Digestion using the BstNI enzyme RET. The 10-wk, three sessions/week RET program included 13 requires 60°C incubation. Presence of the restriction site for the BstNI exercises encompassing all major muscle groups with weights set at or HpaII sites indicated a C allele. Subjects were categorized as 80% of 1 RM. These sessions were supervised by exercise physiolo- exhibiting CC, CA, or AA genotype for BstNI and AA, AG, or GG for gists (participant-to-trainer ratio 2:1 to 3:1). The RET program in- HpaII. Most CC genotypes for the BstNI site corresponded to the AA cluded the following exercises on the Strive (Canonsburg, PA) weight genotype for the HpaII site. Likewise, CA and AA (BstNI) corre- sponded to AG and GG (HpaII), respectively. Therefore, C:A and A:G machines: chest press, seated row, lateral pulldowns, leg extensions, (BstNI:HpaII) haplotypes were assigned as well. triceps extension, arm curl, shoulder press, hamstring curl, low back IL-15 protein analysis. Blood samples were collected in 10 ml extension, abdominal crunch, and incline press. Leg press and calf EDTA (Vacutainer) before the first and last RET session after a raises were performed on a Universal leg press machine. A warm-up minimum 8-h fast. A second blood draw was collected immediately consisting of exercise on an aerobic machine for 5 min followed by after (within 5 min) the first and last RET session. Samples were stretching was performed before each day of RET. Subjects performed immediately placed on ice, centrifuged, and stored at Ϫ70°C in 1-ml three sets of 6–10 repetitions for each exercise. Subjects were given aliquots. Plasma samples were assayed for concentrations of IL-15 30 s of rest between each set and 1 min between exercises. Resistance using the QuantiGlo human IL-15 Immunoassay kit method (R&D settings were increased when participants were able to complete 10 Systems, Minneapolis, MN). The detection range for IL-15 is 0.3 to repetitions on a particular set to maintain the relative difficulty at the 30 pg/ml. The inter- and intra-assay coefficients of variation for each same level for all subjects throughout the study. assay were Ͻ10% throughout the assays (mean Ͻ5%). Genetic analysis. Genomic DNA from 12 control samples of Statistical analysis. SPSS software (SPSS, Chicago, IL, version diverse ethnic background were used to search for genetic variation in 11.0) was used for all analyses. One-way ANOVA was used to the IL-15 receptor gene (IL15RA). Primers were selected so as to analyze differences in baseline characteristics by genotype. A ␹2 cover all exonic regions and exon-intron borders using cDNA se- analysis was used to determine whether the frequencies of the geno- quence found in GenBank (accession no. U31628). After amplifica- types were in Hardy-Weinberg equilibrium. Linkage disequilibrium tion of these regions by PCR (35 cycles), fragments were directly and haplotype estimations were determined using the methods outline sequenced by using the dRhodamine ready reaction kit (Perkin-Elmer) by Seltman et al. (20) and Abecasis and Cookson (1). Because of the and analyzed on the ABI Prism model 377 fluorescent sequencer. strong disequilibrium (DЈ) between the BstNI and HpaII sites (DЈϭ Sequences were aligned for comparison by use of Sequencher 3.0 0.875), the common haplotypes of these sites were determined, and (Gene Codes). Variations were confirmed by restriction enzyme each subject’s haplotype combination for these two sites was catego- digestion of amplicons when more than one of the 24 chromosomes rized for analysis. Three haplotype combinations accounted for 79% indicated a sequence variant. Polymorphisms in exon 4, exon 5 of the MERET cohort (A:G/A:G, A:G/C:A, C:A/C:A). Analysis of intron-exon border, and exon 7 were subsequently genotyped in the covariance methods were used to determine differences in muscle MERET cohort. Methods for other regions are reported in supplemen- responses to RET by genotype and haplotype. We used the strategy of tary material (jap.physiology.org/cgi/content/full/00491.2004/DC1). analyzing percent change of dependent variables to adjust for differ- Genomic DNA was extracted from the buffy coat from blood samples ences in body size that would otherwise skew the relative amount of by standard methods (14). The exon 4 variant (C to A) results in muscle mass or strength gain. Other strategies included differences in Thr/Asn amino acid change (SNP accession rs3136617) and was the z scores between baseline and posttraining, z score of the change, genotyped by using the enzyme BstNI. The exon 5 intron-exon border and the difference between the z score of change and z score of the variant (A to G) is 20 bp from the border in intron 5 (SNP accession baseline. All strategies gave remarkably similar results to those rs3136618) and was genotyped by using the enzyme HpaII. The exon reported for percent change except only percent change adjusted for 7 variant (A to C) is in the 3Ј untranslated region (SNP accession gender differences in response (data not shown). The covariates rs2296135) and was genotyped by using the enzyme PstI. To create a gender, RET history, oral contraceptive use, and age, previously PstI restriction site, it was necessary to insert a mismatch in the reported as significantly associated with muscle phenotypes, were also forward primer. used for these analyses (19). Stepwise multiple regression analyses

J Appl Physiol • VOL 97 • DECEMBER 2004 • www.jap.org 2216 IL15RA POLYMORPHISM AND RESISTANCE TRAINING were performed to test the independent association of IL15RA geno- Table 2. Association of the interleukin-15 receptor PstI types on lean mass response to RET. A repeated-measures ANOVA genotype with muscle phenotype responses to resistance was used to examine differences in IL-15 protein at the beginning and exercise training end of training and response to acute RET. Change in hematocrit was used as a covariate to control for plasma volume shifts with exercise. CC CA AA Pearson’s correlation coefficients were calculated to estimate the association of IL-15 protein concentrations to muscle responses to Lean mass gain, kg 0.8Ϯ0.2 1.6Ϯ0.2 2.0Ϯ0.3* Ϯ Ϯ Ϯ RET. Lean mass gain, % 1.3 0.4 2.9 0.3 3.8 0.5* ϫ Ϫ Arm circ gain, % 3.0Ϯ0.5 4.2Ϯ0.4 5.1Ϯ0.7* Percent gains were calculated as 100 (postmeasurement Leg circ gain, % 0.1Ϯ0.5 1.3Ϯ0.4 2.2Ϯ0.7* premeasurement)/premeasurement. Arm strength was calculated as 1 Arm strength gain, % 49Ϯ344Ϯ342Ϯ4 RM biceps kg ϩ 1 RM triceps kg. Arm muscle quality (MQ) was Leg strength gain, % 41Ϯ339Ϯ236Ϯ4 calculated as arm MQ ϭ (1 RM biceps kg ϩ 1 RM triceps kg)/arm Arm MQ gain, % 44Ϯ339Ϯ332Ϯ4* circumference (cm). Leg strength was calculated as 1 RM hamstring Leg MQ gain, % 41Ϯ337Ϯ328Ϯ4* ϩ curl kg 1 RM leg extension kg. Composite strength for arm and leg Ϯ ϭ ϩ was reported as summary of strength measures that was consistent for Values are means SE. Arm strength triceps extension biceps curl 1 repetition maximum (1 RM), leg strength ϭ hamstring curl ϩ leg extension 1 all measures of strength in this study and specifically in this report RM. Arm muscle quality (MQ) ϭ (1 RM biceps ϩ 1 RM triceps)/arm because these composite scores were used in the calculation to circumference. Leg MQ ϭ (1 RM hamstring curl ϩ 1 RM leg extension)/thigh estimate muscle quality. Leg muscle quality was calculated as leg circumference. *P Ͻ 0.05 for genotype (analysis of covariance). MQ ϭ [(1 RM hamstring curl kg ϩ 1 RM leg extension kg)/(thigh circumference cm)]. Haplotypes. Haplotype frequencies for the IL15RA BstNI, ϭ RESULTS HpaII, and PstI sites were as follows: C:A:C 0.355, A:G: A ϭ 0.296, A:G:C ϭ 0.138, C:A:A ϭ 0.128, A:A:C ϭ 0.025, Subjects. The baseline characteristics stratified by IL15RA A:A:A ϭ 0.022, C:G:C ϭ 0.022, and C:G:A ϭ 0.014. The PstI genotype of the 76 men and 77 women who completed the frequencies of the BstNI and HpaII C:A and A:G haplotypes training are presented in Table 1. Of these subjects, 14 (9.3%) were 0.483 and 0.434, respectively. These BstNI and HpaII of those who completed the training were not Caucasian (i.e., haplotypes were used to analyze muscle responses stratified by African American, Hispanic, Asian). Baseline characteristics PstI genotypes (Table 3). The haplotype combination across all were similar between genotype groups (P Ͼ 0.05) including three sites can be deduced from the table with the exception of HpaII and BstNI genotypes (data not shown). Frequencies of AC/GA/CA (BstNI/HpaII/PstI genotypes), which would have the genotypes were in Hardy-Weinberg equilibrium and were either A:G:C/C:A:A or A:G:A/C:A:C haplotype combination. not different between gender or race categories (P Ͼ 0.05). Muscle mass response and BstNI, HpaII haplotype. The PstI genotype and muscle responses to RET. Changes in presence of a C:A (BstNI:HpaII) haplotype is associated with muscle phenotypes by IL15RA PstI genotype are presented in a significant (P Ͻ 0.05) increase in lean mass (Table 3). The Table 2. Significantly greater increases in total lean mass and addition of a second C:A had no effect on the change in lean arm and leg circumference were observed in those with an A mass. Because data were excluded that did not have the A:G or allele (P Ͻ 0.05). A significant linear trend was observed with C:A haplotypes exclusively or that did not have all three the addition of each A allele for these related phenotypes (P Ͻ genotypes, the sample size was reduced to 118. The effect of 0.05). These observations were significant in both men and the PstI genotypes remained consistent between haplotypes women when analyzed separately (data not shown). However, (i.e., there were no interactions). No additional effects of the arm and leg strength gain was not significantly different be- haplotypes were observed for other muscle response pheno- tween genotype groups (P Ͼ 0.05). The nonsignificant trend types (data not shown). was in the opposite direction, in which the mean relative Multiple regression. To determine the independent associa- strength gain was lower with the addition of each A allele. Arm tion of the IL15RA genotypes on muscle mass response to and leg muscle quality changes, the composite measure of RET, stepwise multiple regression was performed (Table 4). mass and strength gain, were significantly reduced with the PstI and BstNI genotypes were independently associated with addition of each A allele (P Ͻ 0.05). muscle mass response to RET and predicted 7.1 and 3.5% of the gain, respectively. The BstNI/HpaII haplotype variable did not enter the model when BstNI genotypes were submitted as well. Interactions between genotypes and covariates were also Table 1. Subject baseline characteristics by interleukin-15 receptor PstI genotype examined but did not significantly add to the model. Only the PstI site was also independently associated with arm and leg Genotype circumference and muscle quality gains after adjusting for known covariates (data not shown). CC CA AA IL-15 protein. Resting/fasting plasma concentrations of the Men/women (n) 24/22 38/36 14/19 IL-15 protein were significantly increased in response to acute Age, yr 21.1Ϯ2.4 20.9Ϯ2.6 20.7Ϯ2.4 resistance exercise at the first and last (30th) session of the 10 Height, cm 171.6Ϯ9.6 170.3Ϯ8.4 169.5Ϯ9.7 Weight, kg 74.7Ϯ15.0 72.3Ϯ15.0 72.7Ϯ16.3 wk of RET, even after adjustment for plasma volume shifts Fat, % 23.1Ϯ6.1 22.3Ϯ7.7 23.4Ϯ8.8 (Fig. 1). No changes were observed in resting/fasting concen- Lean mass, kg 57.1Ϯ12.1 55.9Ϯ11.4 55.3Ϯ12.4 trations nor in acute responsiveness from the first to the last Values are means Ϯ SD. P Ͼ 0.05 for all comparisons. Frequencies of training session. There were no differences observed for age or genotypes are in Hardy-Weinberg equilbrium (P Ͼ 0.05, p ϭ 0.54, q ϭ 0.46) gender for resting concentrations or responsiveness (data not and are similar between men and women and between black and white. shown); however, African-American participants had signifi-

J Appl Physiol • VOL 97 • DECEMBER 2004 • www.jap.org IL15RA POLYMORPHISM AND RESISTANCE TRAINING 2217 Table 3. Association of the interleukin-15 receptor BstNI/HpaII haplotype and PstI genotype with change in lean mass

IL15R BstNI/HpaII

Haplotype sequence A:G, A:G A:G, C:A C:A, C:A BstNI/HpaII genotype AA/GG N AC/GA N CC/AA NN IL15R PstI CC 0.9Ϯ1.3 5 1.8Ϯ0.9 11 1.5Ϯ0.7 18 1.4Ϯ0.6 34 CA 1.8Ϯ1.3 5 3.1Ϯ0.4 44 3.5Ϯ0.9 10 2.8Ϯ0.5 59 AA 3.3Ϯ0.8 15 4.4Ϯ1.7 3 4.9Ϯ1.1 7 4.2Ϯ0.7 25 2.0Ϯ0.7 25 3.1Ϯ0.7* 58 3.3Ϯ0.5* 35 3.3Ϯ0.5 118 Values are means Ϯ SE (in kg). *P Ͻ 0.05 vs. A:G, A:G cantly greater resting IL-15 concentrations than Caucasians disequilibrium with a functional site. Examination of IL15RA (AA ϭ 2.1 Ϯ 0.2, n ϭ 11, CA ϭ 1.6 Ϯ 0.1, n ϭ 112, P Ͻ variation in a SNP database (http://snpper.chip.org) did not 0.05). These differences did not affect the observed acute reveal any additional variation that would likely alter function changes in IL-15. Resting concentrations and acute and chronic of the receptor. However, alternate exon 7 splicing has been changes in IL-15 were not significantly correlated to muscle observed to cause two distinct exon 7 isoforms within the mass, strength, or quality at baseline or in response to RET IL-15 receptor protein, leading to the possibility that genetic (data not shown). The sample size of these analyses was variation in the IL-15 receptor mRNA influences the splicing reduced because of the lack of availability of all four blood pathway or translation efficiency. The IL-15 receptor has two samples for several subjects. IL-15 protein concentrations were other splice variants in which exon 2 and exon 3 are deleted, not associated with IL15RA genotype. resulting in eight observed isoforms. In the original description of these splice variants, no differences in signaling or binding DISCUSSION of IL-15 were observed (2, 9), with the exception of the exon IL-15 has been shown to be a strong stimulant for muscle 2 deletion isoform, which does not bind IL-15. The exact role growth in animal and cell culture models (6, 17, 18). We of the PstI variation remains to be determined by functional examined the physiological changes of IL-15 in plasma and studies of the gene and protein. No measurements of IL-15 genetic variation in the IL-15 receptor-␣ to determine whether receptor were made in the present study, so no conclusions these factors were associated with the variability in muscle about the potential influence of IL15RA polymorphisms on mass, strength, and quality responses to standardized RET. receptor isoforms can be proposed. Plasma IL-15 increased significantly in response to acute The exon 4 BstNI Asn to Thr variation would seem to be the resistance exercise, but this response did not change with the most interesting because it leads to an amino acid change to 10 wk of RET and was not associated with variability in Thr in a Pro-Thr-rich area of the extracellular domain. How- muscle responses to training. However, the exon 7 PstI C to A ever, the effects on muscle responses to RET were quite polymorphism in the IL-15 receptor-␣ gene was associated modest compared with the PstI site. Although these two sites Јϭ Ͻ with greater muscle mass gains but reduced muscle quality were in modest linkage disequilibrium (D 0.402, P gains. Moreover, IL15RA exon 4 (BstNI) genotypes or BstNI/ 0.001), their effects were independent in regression modeling. HpaII haplotypes were also significantly associated with lean This variation has been reported previously, but these authors mass gain independent of the PstI variation. did not demonstrate differences in cell proliferation or IL-15 The IL15RA PstI genetic variation is found in exon 7 binding (2). This BstNI polymorphism was in very strong Јϭ Ͻ (cytoplasmic-signaling domain) in the 3Ј untranslated region of linkage disequilibrium with the HpaII site (D 0.875, P the gene. The fact that the variation is not translated suggests 0.001). Analysis of the haplotypes of these two sites demon- that this variation may not be responsible for the observed strated a small effect with the addition of one C:T (BstNI: differences in muscle responses and is merely in linkage HpaII) variant. The IL15R HpaII variation is found at the

Table 4. Multiple regression analysis to determine the independent association of IL15 receptor genotypes on lean mass response to 10 wk of RET

Non- Standardized Regression Standard P R2 Coefficient Error Value Change DV ϭ change in lean mass, kg Constant 4.299 1.311 0.001 IL15 receptor PstI genotype 0.810 0.198 Ͻ0.001 0.071 Age, yr Ϫ0.136 0.056 0.017 0.043 OC use Ϫ0.460 0.165 0.006 0.039 RET history, h/wk Ϫ0.221 0.084 0.009 0.042 IL15 receptor BstN1 genotype Ϫ0.470 0.197 0.018 0.035 Fig. 1. Plasma IL-15 protein concentration before and after resistance exercise RET, resistance exercise training. Oral contraceptive (OC) use scores: at the beginning (session 1) and end (session 30) of the resistance exercise Men ϭ 0, No OC ϭ 1, OC ϭ 2. training intervention (N ϭ 124).

J Appl Physiol • VOL 97 • DECEMBER 2004 • www.jap.org 2218 IL15RA POLYMORPHISM AND RESISTANCE TRAINING intron/exon border of exon 5 (intron 5). It is difficult to The effects of TNF-␣ may also play an important role in distinguish from these analyses which polymorphism exerts the IL-15-mediated hypertrophy. TNF-␣ is a catabolic cytokine effect because of the disequilibrium, but it is likely that the and has been shown to be elevated in muscle inflammatory BstNI site is responsible owing to the amino acid change and disease (23, 25), muscle injury models (8), cachexia (7, 26), consistency of the BstNI variant in muscle response analyses. and frail elderly and is reduced with resistance training in the An unexpected finding was the lower muscle quality re- frail elderly (12). Higher levels of TNF-␣ were also associated sponses in the AA PstI genotype group compared with the CA with lower muscle mass in elderly (28). TNF-␣ stimulates an and CC groups. The PstI AA group demonstrated the greatest important pathway in apoptosis signaling (29), whereas IL-15 hypertrophic response to RET yet the lowest increase in can potently inhibit the process (5). Moreover, the IL-15 strength (P Ͼ 0.05). Hypertrophy has been considered the ideal receptor can specifically block TNF-␣-mediated apoptosis by response to resistance training and cellular studies of muscle recruiting the TNF receptor-associated factor (TRAF2) and growth, but muscle quality has been examined presently to preventing TNF signaling. This may be accomplished by CD40 reflect a measure of mass to performance efficiency. If neuro- TRAF2 interacting motifs that are also found in the cytoplas- logical and biochemical adaptations are not sufficient to re- mic tail of the IL15R protein (4). IL-15 receptor signaling may spond to the overload stimulus, hypertrophy may be a com- also rescue cells from apoptosis through phosphorylation of pensating adaptation. This is supported by the observation that I␬B and subsequent activation of NF-␬B. It may be possible early adaptations to RET are neurological and biochemical in that the genetic variation in the IL-15 receptor affects this nature, followed later by hypertrophy. particular mechanism of IL-15 action on muscle hypertrophy IL-15 is heavily concentrated in skeletal muscle (11, 17, 15). directly or that isoforms of the IL-15 receptor protect against Two isoforms of IL-15 have been observed and either remain TNF-mediated apoptosis to different degrees. intracellularly in the cytoplasm and nucleus (SSP-IL-15) or In summary, chronic RET induces skeletal muscle hypertro- have no nuclear localization and are directed to secretory phy as well as increases in strength. However, not every pathways (LSP-IL-15) (24). In the report by Tagaya et al. (24), individual can expect the same magnitude of muscle responses only the secreted LSP-IL-15 isoform mRNA is found in mon- to a standard program because of genetic and environmental key skeletal muscle. Subsequent reports have demonstrated factors yet to be thoroughly characterized. The effect of IL-15 both isoforms of the IL-15 protein present in the SkMC 2859 signaling was examined as a contributing mediator of this human skeletal muscle cell line (23). Although plasma con- interindividual variability in muscle responses by measuring centrations increased significantly with acute exercise, vari- IL-15 protein in plasma during exercise and training and ability in the IL-15 levels and responses were not associated characterizing genetic variation in the IL15RA gene. Plasma with muscle responses to training. Alternately, it has been IL-15 was increased immediately after exercise but did not demonstrated that IL-15 plays a role in angiogenesis (3). Acute change with training and was not associated with muscle changes in IL-15 with exercise may be associated with the responses to training. A genetic variation in the 3Ј untranslated changes in blood supply requirements and neovascularization region of the IL15RA gene was strongly associated with that occur with training. Therefore, study of the physiological muscle hypertrophy, although those with the greatest hypertro- role of IL-15 in skeletal muscle response to acute exercise may phy had lower muscle strength and muscle quality increases. require direct assessment of IL-15 intramuscularly. Another genetic variation that resulted in the addition of a Thr If the effects of IL-15 on muscle are primarily local, a in the Pro- and Thr-rich area of the IL-15 receptor was weakly possible mechanism may be the effect of IL-15 on immune associated with muscle hypertrophy. Taken together with other function. The immune inflammatory response is widely be- studies of IL-15 on muscle anabolism, this study suggests that lieved to play an important role in skeletal muscle hypertrophy IL-15 is an important mediator of muscle phenotypes in hu- (27). Microtears in skeletal muscle with acute resistance exer- mans. cise result in the recruitment of neutrophils, macrophages, and lymphocytes to muscle and a subsequent inflammatory re- ACKNOWLEDGMENTS sponse. Cytokines and growth factors released as a result of We extend gratitude to the undergraduate and graduate students (especially this response contribute to repairing damaged tissue and hyper- Ryan Witt) in the Exercise Physiology program at the University of Pittsburgh trophy. with whom this project has been successfully completed. IL-15 is well known to stimulate activation and proliferation of the IFN-␥-producing T cells and natural killer cells and to GRANTS ␥ stimulate antibody synthesis in B cells. IFN- has been shown This work is supported by the Obesity and Nutrition Research Center to induce cell surface presentation of the CD40 protein on Pilot/Feasibility Grant P30-DK046204 (S. E. Riechman), a Developmental and human myocytes (SkMC 2859), which on interaction with Feasibility Project for the Multipurpose Arthritis and Skeletal Diseases Center CD40 ligand (CD40L) from T-cells stimulates IL-15 produc- (MAMDC) Grant 5 P60 AR44811-04 SUB: 0007 (S. E. Riechman, R. E. tion in these myocytes (23). In another study, IL-4 stimulated Ferrell), and National Institutes of Health Grants DK-45204 (R. E. Ferrell) and AG-05893 and AG-022791 (S. M. Roth). IL-15 production in primary skeletal muscle cell line (LE1) (22). Because IL-15 can stimulate IFN-␥ producing cells, Stegall and Krolick (22) also demonstrated that IFN-␥ modu- REFERENCES lates the IL-4 effects on myocyte IL-15 production. Moreover, 1. Abecasis GR and Cookson WO. GOLD—graphical overview of linkage Horsley et al. (13) demonstrated that myotubes recruit myo- disequilibrium. Bioinformatics 16: 182–183, 2000. 2. Anderson DM, Kumaki S, Ahdieh M, Bertles J, Tometsko M, Loomis blast fusion by secretion of IL-4 as an important component of A, Giri J, Copeland NG, Gilbert DJ, Jenkins NA, Valentine V, muscle hypertrophy, although it remains to be determined Shapiro DN, Morris SW, Park LS, and Cosman D. Functional charac- whether IL-15 plays a role in these observations. terization of the human interleukin-15 receptor alpha chain and close

J Appl Physiol • VOL 97 • DECEMBER 2004 • www.jap.org IL15RA POLYMORPHISM AND RESISTANCE TRAINING 2219

linkage of IL15RA and IL2RA genes. J Biol Chem 270: 29862–29869, skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h 1995. run. J Appl Physiol 94: 1917–1925, 2003. 3. Angiolillo A, Kanegane H, Sgadari C, Reaman GH, and Tosato G. 16. Quinn LS, Anderson BG, Drivdahl RH, Alvarez B, and Argiles JM. Interleukin-15 promotes angiogenesis in vivo. Biochem Biophys Res Overexpression of interleukin-15 induces skeletal muscle hypertrophy in Commun 233: 231–237, 1997. vitro: implications for treatment of muscle wasting disorders. Exp Cell Res 4. Bulfone-Paus S, Bulanova E, Pohl T, Budagian V, Durkop H, Ruckert 280: 55–63, 2002. R, Kunzendorf U, Paus R, and Krause H. Death deflected: IL-15 17. Quinn LS, Haugk KL, and Damon SE. Interleukin-15 stimulates C2 inhibits TNF-␣-mediated apoptosis in fibroblasts by TRAF2 recruitment skeletal myoblast differentiation. Biochem Biophys Res Commun 239: to the IL-15R␣ chain. FASEB J 13: 1575–1585, 1999. 6–10, 1997. 5. Bulfone-Paus S, Ungureanu D, Pohl T, Lindner G, Paus R, Ruckert R, 18. Quinn LS, Haugk KL, and Grabstein KH. Interleukin-15: a novel Krause H, and Kunzendorf U. Interleukin-15 protects from lethal apop- anabolic cytokine for skeletal muscle. Endocrinology 136: 3669–3672, tosis in vivo. Nat Med 3: 1124–1128, 1997. 1995. 6. Carbo´ N, Lopez-Soriano J, Costelli P, Busquets S, Alverez B, Baccino 19. Riechman SE, Fabian TJ, Kroboth PD, and Ferrell RE. Steroid FM, Quinn LS, Lopez-Soriano FJ, and Argiles JM. Interleukin-15 sulfatase gene variation and DHEA responsiveness to resistance exercise antagonizes muscle protein waste in tumor-bearing rats. Br J Cancer 83: in MERET. Physiol Genomics 17: 300–306, 2004. 526–531, 2000. 20. Seltman H, Roeder K, and Devlin B. Evolutionary-based association 7. Coletti D, Yang E, Marazzi G, and Sassoon D. TNF␣ inhibits skeletal analysis using haplotype data. Genet Epidemiol 25: 48–58, 2003. myogenesis through a PW1-dependent pathway by recruitment of caspase 21. Siri WE. Body composition from fluid spaces and density; analysis of pathways. EMBO J 21: 631–642, 2002. methods. In: Techniques for Measuring Body Composition, edited by 8. Collins RA and Grounds MD. The role of tumor necrosis factor-alpha Brozek J and Henschel A. Washington, DC: Natl. Acad. Sci., 1961, p. (TNF-␣) in skeletal muscle regeneration. Studies in TNF-␣(Ϫ/Ϫ) and 223–244. 22. Stegall T and Krolick KA. Myocytes respond to both interleukin-4 and TNF-␣(Ϫ/Ϫ)/LT-␣(Ϫ/Ϫ) mice. J Histochem Cytochem 49: 989–1001, interferon-gamma: cytokine responsiveness with the potential to influence 2001. the severity and course of experimental myasthenia gravis. Clin Immunol 9. Dubois S, Magrangeas F, Lehours P, Raher S, Bernard J, Boisteau O, 94: 133–139, 2000. Leroy S, Minvielle S, Godard A, and Jacques Y. Natural splicing of 23. Sugiura T, Kawaguchi Y, Harigai M, Takagi K, Ohta S, Fukasawa C, exon 2 of human interleukin-15 receptor alpha-chain mRNA results in a Hara M, and Kamatani N. Increased CD40 expression on muscle cells J Biol Chem shortened form with a distinct pattern of expression. 274: of polymyositis and dermatomyositis: role of CD40-CD40 ligand interac- 26978–26984, 1999. tion in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 produc- 10. Giri JG, Anderson DM, Kumaki S, Park LS, Grabstein KH, and tion. J Immunol 164: 6593–6600, 2000. Cosman D. IL-15, a novel T-cell growth factor that shares activities and 24. Tagaya Y, Kurys G, Thies T, Losi JM, Azimi N, Hanover JA, receptor components with IL-2. J Leukoc Biol 57: 763–766, 1995. Bamford RN, and Waldmann TA. Generation of secretable and non- 11. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, secretable interleukin 15 isoforms through alternate usage of signal pep- Fung V, Beers C, Richardson J, Schoenborn MA, Ahdieh M, Johnson tides. Proc Natl Acad Sci USA 94: 14444–14449, 1997. L, Alderson MR, Watson JD, Anderson DM, and Giri JG. Cloning of 25. Tateyama M, Nagano I, Yoshioka M, Chida K, Nakamura S, and a T cell growth factor that interacts with the beta chain of the interleukin-2 Itoyama Y. Expression of tumor necrosis factor-␣ in muscles in polymy- receptor. Science 264: 965–968, 1994. ositis. J Neurol Sci 146: 45, 1997. 12. Greiwe JS, Cheng B, Rubin DC, Yarasheski KE, and Semenkovich 26. Tisdale MJ. Loss of skeletal muscle in cancer: biochemical mechanisms. CF. Resistance exercise decreases skeletal muscle tumor necrosis factor ␣ Front Biosci 6: D164–D174, 2001. in frail elderly humans. FASEB J 15: 475–482, 2001. 27. Vierck J, O؅Reilly B, Hossner K, Antonio J, Byrne K, Bucci L, and 13. Horsley V, Jansen KM, Mills ST, and Pavlath GK. IL-4 acts as a Dodson M. Satellite cell regulation following myotrauma caused by myoblast recruitment factor during mammalian muscle growth. Cell 113: resistance exercise. Cell Biol Int 24: 263–272, 2000. 483–494, 2003. 28. Visser M, Pahor M, Taaffe DR, Goodpaster BH, Simonsick EM, 14. Miller SA, Dykes DD, and Pulesky HF. A simple salting out procedure Newman AB, Nevitt M, and Harris TB. Relationship of interleukin-6 for extracting DNA from human nucleated cells. Nucleic Acids Res 16: and tumor necrosis factor-alpha with muscle mass and muscle strength in 1215, 1988. elderly men and women: the Health ABC Study. J Gerontol A Biol Sci 15. Nieman DC, Davis JM, Henson DA, Walberg-Rankin J, Shute M, Med Sci 57: M326–M332, 2002. Dumke CL, Utter AC, Vinci DM, Carson JA, Brown A, Lee WJ, 29. Wallach D. Cell death induction by TNF: a matter of self control. Trends McAnulty SR, and McAnulty LS. Carbohydrate ingestion influences Biochem Sci 22: 107–109, 1997.

J Appl Physiol • VOL 97 • DECEMBER 2004 • www.jap.org