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doi: 10.1152/japplphysiol.00817.200498:2147-2154, 2005. ;J Appl Physiol

Douglass, Robert E. Ferrell and Ben F. HurleyMatthew C. Kostek, Matthew J. Delmonico, Jonathan B. Reichel, Stephen M. Roth, Larryolder adultsinfluenced by insulin-like growth factor 1 genotype inMuscle strength response to strength training is

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Muscle strength response to strength training is influenced by insulin-like

growth factor 1 genotype in older adults

Matthew C. Kostek,1 Matthew J. Delmonico,1 Jonathan B. Reichel,1

Stephen M. Roth,1 Larry Douglass,1 Robert E. Ferrell,2 and Ben F. Hurley1

1Department of Kinesiology, College of Health and Human Performance, University of Maryland, College Park, Maryland;and 2Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 30 July 2004; accepted in final form 27 December 2004

Kostek, Matthew C., Matthew J. Delmonico, Jonathan B. Reichel,Stephen M. Roth, Larry Douglass, Robert E. Ferrell, and Ben F.Hurley. Muscle strength response to strength training is influenced byinsulin-like growth factor 1 genotype in older adults. J Appl Physiol 98:21472154, 2005; doi:10.1152/japplphysiol.00817.2004.Strength train-ing (ST) is considered an intervention of choice for the prevention andtreatment of sarcopenia. Reports in the literature have suggested thatthe insulin-like growth factor I protein (IGF-I) plays a major role inST-induced skeletal muscle hypertrophy and strength improvements.A microsatellite repeat in the promoter region of the IGF1 gene hasbeen associated with IGF-I blood levels and phenotypes related toIGF-I in adult men and women. To examine the influence of thispolymorphism on muscle hypertrophic and strength responses to ST,we studied 67 Caucasian men and women before and after a 10-wksingle-leg knee-extension ST program. One repetition maximumstrength, muscle volume via computed tomography, and musclequality were assessed at baseline and after 10 wk of training. TheIGF1 repeat promoter polymorphism and three single-nucleotidepolymorphisms were genotyped. For the promoter polymorphism,subjects were grouped as homozygous for the 192 allele, heterozy-gous, or noncarriers of the 192 allele. After 10 wk of training,1-repetition maximum, muscle volume, and muscle quality increasedsignificantly for all groups combined (P 0.001). However, carriersof the 192 allele gained significantly more strength with ST than

noncarriers of the 192 allele (P 0.02). There was also a nonsignif-icant trend for a greater increase in muscle volume in 192 carriers thannoncarriers (P 0.08). No significant associations were observed forthe other polymorphisms studied. Thus these data suggest that theIGF1 promoter polymorphism may influence the strength response toST. Larger sample sizes should be used in future studies to verifythese results.

muscle volume; muscle quality

HUMAN MUSCLE STRENGTH DECLINES at the rate of1214% perdecade after the age of50 yr (31, 33). This loss of strengthwith age is due to many factors but is primarily attributed to aloss of muscle mass (21, 42), termed sarcopenia. Because

sarcopenia is related to a loss of functional abilities (39),dependency (40), increased risk of falls and fractures (4, 32),and decreased bone mineral density (51), it has negativeconsequences for the health status and functional abilities ofolder adults.

Several interventions have been proposed for the preven-tion and treatment of sarcopenia, but it appears that strengthtraining (ST) is the most effective with the least negativeside effects (7, 25). Large increases in strength and muscle

mass can result in a relatively short time with ST, but thesechanges are highly variable (26). For example, in a ST studyfrom our laboratory (26, 30), 65- to 75-yr-old men andwomen varied in their strength increases from 5 to 59%(586 lb.) and muscle volume (MV) increases from 1 to20% (19344 ml). This variation in muscle adaptation totraining may suggest a genetic influence. Further support forgenetic associations come from twin studies showing that50% of the variance in baseline strength and lean body

mass is heritable (9, 19, 38, 50). Adaptation of muscle to SThas also been shown to be highly heritable (53, 54). Yet,there is little information on how specific polymorphismsmay affect this response.

Circulating levels of insulin-like growth factor 1 protein(IGF-I) decline with advancing age, and IGF-I is thought to becausally related to the loss of muscle mass and strength thatoccurs with age (23, 56). Furthermore, exogenous growthhormone administration increases circulating IGF-I levels,which have been shown to increase muscle mass and possiblystrength (12, 49). Yet, it seems that circulating levels of IGF-Iare not as important for muscle growth as are the isoforms ofIGF-I produced by skeletal muscle, which act in an autocrine/paracrine fashion (1, 5, 22). Studies with transgenic mice haveshown a direct autocrine/paracrine effect of increased levels ofIGF-I expression increasing skeletal muscle mass (17). Thissame transgenic line of mice, compared with controls, showsreduced age-related losses of skeletal muscle motoneurons andtype IIB muscle fibers (35). This preservation of motoneuronsand fast-twitch fibers is likely to preserve strength in agingmuscle. In addition, Boonen et al. (8) observed an increase inmuscle strength in older women after recombinant humanIGF-I administration.

Due to their known association with muscle hypertrophy,genes coding for proteins that regulate or are regulated bygrowth hormone or its mediators (IGF-I and -II) are primecandidate genes for influencing muscle mass and strength.

Indeed, a report from the HERITAGE Family Study revealedan association between an IGF1 gene marker and baselinefat-free mass (FFM) (11). Furthermore, this same marker hasbeen shown to be linked and associated with the change inFFM due to exercise training (11, 52). However, the trainingmodality used in the HERITAGE study (11) was one that is notcommonly associated with significant increases in FFM. Nev-ertheless, an area near the IGF1 gene locus was implicated inthese studies for influencing FFM both at baseline and in

Address for reprint requests and other correspondence: B. F. Hurley, Dept.of Kinesiology, Univ. of Maryland, College Park, MD 20742 (E-mail:[email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Appl Physiol 98: 21472154, 2005;doi:10.1152/japplphysiol.00817.2004.

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response to training, thus making it a strong candidate gene formuscle mass as well as strength.

Recent studies have shown that a cytosine adenine (CA)dinucleotide repeat polymorphism in the promoter region ofthe IGF1 gene can affect blood levels of IGF-I (20, 29, 36, 44,46, 58, 59). The polymorphism in the IGF1 gene is typicallybetween 16 and 22 CA repeats and is commonly referred to by

the base pair length of the amplified DNA fragment (e.g., 192bp). Homozygosity for the 192 allele (19 CAs at nucleotideposition 1,0871,127 in the human IGF1 DNA sequence Gen-bank accession number AY260957) has been associated withdifferences in circulating levels of IGF-I and phenotypes re-lated to IGF-I (20, 29, 36, 44, 46, 58). Although the function-ality, if any, of this polymorphism remains unknown, it appearsthat it may be associated with the functional properties of andphenotypes related to IGF1. To date, the tissues studied inrelation to this polymorphism have been of the type to beprimarily affected by the endocrine action of IGF-I. Skeletalmuscle, however, is known to be affected by the autocrine/paracrine action of IGF-I, making it a unique tissue in thisrespect. Yet, even with the known effects of IGF-I on muscle,this polymorphism has never been studied in relation to skel-etal muscle phenotypes.

It is possible that the 192 polymorphism is in linkagedisequilibrium with another functional polymorphism, thusresulting in the associations seen in the aforementioned studies.Although no nonsense, missense, or functional polymorphismshave been identified for the IGF1 gene (41), recently identifiedpolymorphisms in IGF1 may serve as useful candidate markersfor phenotypes related to expression levels of IGF1. Because ofthe relationships described above between a polymorphism inIGF1 and expression levels of the IGF-I protein and betweenthe IGF-I protein and muscle function and muscle motoneu-rons, we hypothesize that IGF1 genotype will influence skel-

etal muscle mass and strength response to ST. Due to theknown adverse health consequences of sarcopenia and thereported decline in IGF1 expression in the elderly, we soughtto examine this hypothesis in older adults.

Although there are preliminary data from our laboratory tosupport this hypothesis, no published studies have examinedthis relationship. Understanding this relationship could even-tually lead to individualized exercise prescriptions for olderadults. Therefore, it is the purpose of this study to determinewhether IGF1 genotype in the promoter region or in threerecently identified single-nucleotide polymorphisms (SNPs)within the IGF1 locus influence muscle strength and hypertro-phy in response to ST in middle-aged and older Caucasian menand women.

METHODS

Subjects. Healthy, inactive Caucasian men (n 32) and women(n 35) volunteers between the ages of 52 and 83 yr were recruitedthrough local senior newspaper advertisements and bulk mailings. Aportion of the subjects (n 18) in this investigation are from a cohortthat had already been studied in our laboratory before this study (26).All subjects from both cohorts underwent a phone-screening inter-view, received medical clearance from their primary care physician,and completed a detailed medical history before participating in thisstudy. Only those who had not exercised more than once a weekduring the 6 mo before the study were allowed to participate. Allsubjects from both cohorts were nonsmokers and were free of signif-

icant cardiovascular, metabolic, or musculoskeletal disorders thatwould affect their ability to safely perform heavy resistance exercise.After all methods and procedures, which were approved by theInstitutional Review Board of the University of Maryland, CollegePark, were explained, the subjects read and signed a written consentform. All subjects were reminded throughout the study not to altertheir regular physical activity levels or dietary habits for the durationof the investigation.

Genotyping of CA microsatellite within the IGF1 promoter.Genomic DNA was prepared from EDTA-anticoagulated whole bloodsamples by standard salting-out procedures (Puregene DNA Extrac-tion, Gentra Systems). The microsatellite was amplified by PCR ofgenomic DNA using fluorescence-tagged primers. Previously pub-lished PCR primers (46) flanking the CA polymorphism were usedwith the forward primer FAM-labeled 5-GCTAGCCAGCTGGTGT-TATT-3 (sense) and 5-ATGGGAAGAGGGTCTCACCA-3 (anti-sense).

Standard PCRs were performed on a PTC-100 Thermal Cycler (MJResearch), annealing steps of 1.0 min at 37C, and elongation steps of1.0 min at 72C for 35 cycles were standardized for PCR reactions.All PCR reactions were carried out in a 25-l volume containing 20ng of genomic DNA template: 14.15 ml of dH2O, 2.5 ml of 10 PCRbuffer, 4.0 ml of 1.25 mM dNTP, 0.375 ml of forward primer (20

M), 0.375 ml of reverse primer (20 M), 0.1 ml of Taq DNApolymerase (5 U/ml), and 1.5 ml of 25 mM MgCl2. After amplifica-tion, samples were diluted with distilled, deionized water and mixedwith deionized formamide and 1 U internal size standard labeled withROX fluorescent dye (Genescan-500, PE Applied Biosystems) andthen denatured at 90C for 2 min and immediately transferred ontoice. Samples were electrophoresed through a capillary on the ABI3100 DNA sequencer (PE Applied Biosystems). The ABI Genescan/Genotyper 2.5 software programs (PE Applied Biosystems) were usedto determine repeat length of each microsatellite by comparison withthe calibration curve of the internal standard. Genotypes of threehomozygotes were verified via direct sequencing on the ABI 3100.Additionally, positive controls were used during the sequencing runs.As previously reported, use of the forward primer results in the PCRproduct appearing four bases shorter than actual size; therefore, a

correction factor was applied (46). Briefly, use of the forward primerresulted in PCR bands that moved through the gel at the rate of a band4 bp shorter than actual size. This was confirmed by direct sequencingand was consistent throughout all samples. Therefore, four bases wasadded to the genotyping of each PCR product (i.e., 188 4 192,which 19 CA repeats).

Subject genotypes are based on the PCR fragment length, whichrepresents the actual number of CA repeats present at this locus,typically 1622. The convention for reporting the genotype of theIGF1 promoter is based on the PCR fragment length, which wasvariable depending on the number of repeats (e.g., 19 CA repeats 192 base pairs) (46). Thus PCR base pair length number was reportedto remain consistent with existing conventions.

PCR and RFLP genotyping for SNPs. All PCR reaction mixtureswere run under similar conditions as those stated for the microsatellite

PCR with the exception of annealing temperatures. All primers weredesigned based on SNP location and ordered from IDT DNA tech-nologies. Primers and annealing temperatures are listed in Table 1. AllPCR amplification checks were performed in 2% agarose gels untiloptimized.

Restriction enzymes were selected for each SNP by Webcutter 2.0and purchased from MBI Fermentas. All digests were run accordingto manufacturers recommendations. Restriction enzymes and diges-tion temperatures are listed in Table 1. Briefly, the 19,779 PCRfragment was digested in a 20-l dilution of 2 U of enzyme, 2.0 l ofmanufacturers standard buffer, 2.8 l of dH20, and 15 l of the PCRproduct. All digests were allowed to proceed overnight and were runon a 2% gel after 24 h, with the exception of 40,395, which was runon a 4% gel to achieve optimal separation of bands. Two independent

2148 IGF1 GENOTYPE AND STRENGTH TRAINING

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investigators determined genotypes. Direct sequencing of three PCRproducts from each SNP was performed to verify genotypes.

Body composition assessment. Body weight was determined to thenearest 0.1 kg with subjects dressed in medical scrubs, height wasmeasured to the nearest 0.1 cm using a stadiometer (Harpenden,Holtain, Wales, UK), and BMI was calculated (kg/m2).

Dual-energy X-ray absorptiometry. Body composition was esti-mated by dual-energy X-ray absorptiometry using the fan-beam tech-nology (model QDR 4500A, Hologic, Waltham, MA). A total bodyscan was performed at baseline and again 2448 h after the finalsession of the single-leg ST program. A standardized procedure forpatient positioning, attire, and utilization of the QDR software wasused. Total body FFM, fat mass, and percent fat were analyzed usingHologics version 8.21 software for tissue area assessment. Total bodyFFM is defined as lean soft tissue mass plus total body bone mineralcontent. The coefficients of variation (CV) for all dual-energy X-rayabsorptiometry measures of body composition were calculated fromrepeated scans of 10 subjects who were scanned three consecutivetimes with repositioning (FFM CV 1%; % body fat CV 1.0%).The scanner was calibrated daily against a spine calibration block andstep phantom block supplied by the manufacturer. In addition, a wholebody phantom was scanned weekly to assess any machine drift overtime.

One-repetition maximum strength test. The 1-repetition maximum(1 RM; i.e., the highest resistance at which 1 repetition could besuccessfully completed) strength test was assessed in the knee exten-sors before and after training. Three low-resistance familiarization-training sessions were conducted before baseline 1 RM strength

testing so that subjects would be familiar with the equipment andproper exercise techniques. After the three familiarization trainingsessions and before the regular training sessions began, knee extensor1 RM strength of each leg individually was assessed on the trainingapparatus (Keiser A-300 leg extension machine). After a warm-upconsisting of 23 min of light cycling, subjects were positioned witha pelvis strap (seat belt) to minimize the involvement of other musclegroups. The knee-extension 1 RM test started with a resistance levelestimated to be 2030% of each subjects predicted 1 RM. After60 s of rest on a successful completion of a repetition, subsequenttrials were performed at progressively higher resistance levels tominimize the total number of trials required before the true 1 RMvalue was obtained. Approximately the same number of trials wasused for the after-training test as was used for baseline testing. Allaspects of measuring 1 RM strength before and after 10 wk of

single-leg training were standardized for each subject, includingspecific seat adjustments, body position, and level of vocal encour-agement.

MV. To quantify quadriceps MV, computed tomography (CT)imaging of the trained and untrained thighs was performed (GELightspeed Qxi, General Electric, Milwaukee) at baseline and duringthe last weeks of the 10-wk unilateral ST program. Axial sections ofboth thighs were obtained starting at the most distal point of theischial tuberosity down to the most proximal part of the patella whilesubjects were in a supine position. Measurements of MV in theuntrained leg served as a control for seasonal, methodological, andbiological variation of MV by subtracting the changes in the controlleg from the training-induced changes in the trained leg. Sectionthickness was fixed at 10 mm, with 40 mm separating each section,

based on previous work in our laboratory by Tracy et al. (57).Quadriceps MV was estimated based on using a 4-cm interval be-tween the center of each section. Each CT image was obtained at 120kVp with the scanning time set of 1 s at 40 mA. A 48-cm field of viewand a 512 512 matrix was used to obtain a pixel resolution of 0.94mm. Two technicians performed analyses of all images for eachsubject using Medical Image Processing, Analysis, and Visualizationsoftware (National Institutes of Health, Bethesda, MD). The quadri-ceps cross-sectional area was manually outlined in every 10-mm axialimage from the first section closest to the superior border of the patellato a point where the quadriceps muscle group was no longer reliablydistinguishable from the adductor and hip flexor groups. The same

number of sections proximal from the patella was measured for aparticular subject before and after training to ensure within-subjectmeasurement replication. Investigators were blinded to subject iden-tification, training status, and genotype for both baseline and after-training analysis. Repeated-measurement CV was calculated for eachinvestigator based on repeated measures of selected axial sections ofone subject on 2 separate days. Average intrainvestigator CV was 1.7and 2.3% for investigator 1 and 2, respectively. The average interin-vestigator CV was 3.8%. Final MV was calculated with the use of atruncated cone formula as reported by Tracy et al. (57) and describedby Ross et al. (47). Calculation of muscle quality (MQ) was strength(in N)/MV (in ml). To verify replication of position after training,scout scans were analyzed to verify the ischial tuberosity landmark. Ifthe landmark was found to be 2 cm above or below the most distalpoint of the ischial tuberosity, subjects MV data were not used in any

analysis. Additionally, if a quadricep cross-sectional area was unableto be analyzed due to scan clarity, subjects MV data were not used inany analysis.

Training program. The training program consisted of unilateral(single leg) training of the knee extensor of the dominant leg threetimes per week for 10 wk. Training was performed on a KeiserA-300 air-powered leg-extension machine that allows ease of chang-ing the resistance without interrupting the cadence of the exercise. Theuntrained control leg was kept in a relaxed position throughout thetraining program.

Subjects would warm-up on a bicycle ergometer for 2 min beforeeach training session. The training consisted of five sets of knee-extension exercise for those75 yr of age and four sets for those 75yr of age. The protocol was designed to include a combination ofheavy-resistance and high-volume exercise. The first set was consid-

ered a warm-up and consisted of five repetitions at 50% of the 1 RMstrength value. The second set consisted of five repetitions at thecurrent 5 RM value. The 5 RM value was increased continuallythroughout the training program to reflect increases in strength levels.The first four or five repetitions of the third set were performed at theircurrent 5 RM value, and then the resistance was lowered just enoughto complete one or two more repetitions before muscular fatigue wasreached. This process was repeated until a total of 10 repetitions werecompleted. This same procedure was then used in the fourth and fifthsets, but the total number of repetitions increased in each set. Thefourth set consists of 4 or 5 repetitions at the 5 RM resistance followedby 10 more repetitions for a total of 15 repetitions carried out in thesame manner as described above for the 10-repetition set. The fifth setconsisted of 4 or 5 repetitions at the 5 RM resistance, followed by 15

Table 1. Sequences of PCR primers used for amplification and sequencing of IGF1 gene SNPs

SNP Forward Primer Reverse PrimerAnnealing

Temperature, CRestriction Enzyme/Digestion

Temperature, C

19779 5-CAGATTTGTGGCACTAATAC 5-CTATTAAAGGGATGACTGTG 49 EcoRI/3740395 5-AAGTTAAGAAGCAGTGTTGC 5-AGTGGTGCAATTGTGGCTCA 55 AluI/3782681 5-ACATACAGGTTCTGTGGAAT 5-GTTGGAGAGGATTATGTGTT 51 TaiI/65

Single nucleotide polymorphism (SNP) numbering represents nucleotide position in the human IGF1 DNA sequence. Genbank accession number AY26095.

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more repetitions for a total of 20 repetitions performed in the samemanner as the other sets. This procedure allowed subjects to usenear-maximal effort on every repetition while maintaining a relativelyhigh training volume. The second, third, fourth, and fifth sets arepreceded by rest periods lasting 30, 90, 150, and 180 s, respectively.The shortening phase of the exercise took2 s, and the lengtheningphase took3 s. Subjects performed supervised stretching of the kneeextensors and hip flexors after each training session.

Statistical analyses. All statistical analyses as described belowwere performed using SAS software (SAS version 8.2, SAS Institute,Cary, NC). All analyses were conducted on the change values in thedependent variables. For MV, the change value was calculated bytaking the difference between the change (pre- to posttraining) in MVof the untrained leg and the change in MV (pre- to posttraining) of thetrained leg. For strength, the change in 1 RM value of the trained legwas used as a dependent variable. MQ was calculated as 1 RM/MV,and the change in the trained leg was used as the dependent variableof MQ. The IGF1 promoter polymorphism was categorized as previ-ously reported in the literature as 192 homozygotes, 192 heterozy-gotes, or noncarriers of the 192 allele.

To examine the effect of the promoter genotype on response to ST,an analysis of covariance was used for each dependent variable. Thethree levels of the predefined groupings of genotype at the IGF1

promoter locus were defined as the fixed effect. Covariates wereincluded in the model when significant. Age and gender were includedin all models, and baseline values of the dependent variable wereincluded in each model (i.e., baseline value of 1 RM of the trained legwas the covariate for the analysis of strength). Preplanned compari-sons of 192 carriers vs. noncarriers and 192 homozygotes vs. het-erozygotes and noncarriers were analyzed for each dependent vari-able.

The SNPs were analyzed separately due to a smaller sample sizebecause DNA from 18 subjects was unavailable for SNP genotyping.Due to this smaller sample size and distribution of the three genotypegroups, genotype groups with less than two data points (any genotypewith 1 or no subjects) were not included in the analysis. One 2 2 3 reduced model factorial ANOVA for each of the dependent vari-ables muscle strength and MV was analyzed. The two levels of the

19,779 and 40,395 loci and three levels of the 82,686 loci genotypesform the factorial, and dependent variables were calculated as de-scribed for the promoter analysis. Age, gender, and baseline valueswere included as covariates in the factorial ANOVA as described forthe promoter analysis. Post hoc comparisons were made comparing allmain effects by least significant difference post hoc analysis, andsignificance was accepted when P 0.05 for all analyses.

Hardy-Weinberg equilibrium. The IGF1 genotype distribution wasevaluated for conformity to Hardy-Weinberg equilibrium using a 2

test (degrees of freedom 1).

Power analyses. Statistical power for the three primary compari-sons was estimated for the 192-genotype effect on each variable. Thisanalysis was done using data from a study in our laboratory thatemployed a similar training and testing protocol. Statistical power forchanges in strength a priori was estimated to be 0.80 with set at0.05 (critical effect size MV 0.4; 1 RM 0.45) and accounting forpresent sample size and genotype distribution. However, MV powerwas estimated to be 0.60 due to the smaller training-induced criticaleffect size.

RESULTS

Genotype. IGF1 promoter allele and genotype frequencies(Table 2) did not differ significantly from Hardy-Weinbergexpectations and were similar to those reported previously (36,46, 58). Likewise, IGF1 SNP allele and genotype frequenciesdid not differ significantly from Hardy-Weinberg expectations(Table 3).

Subject characteristics. One female subjects 1 RM valuefrom the 192 homozygote group was lost before data analysis;therefore, her strength values were not included in any analy-sis. Due to stringent CT scan inclusion criteria, seven subjectswere not included in CT analysis [four 192 homozygotes (3women and 1 man) and three 192 heterozygotes (2 women and1 man)]. Body mass, percent body fat, and FFM for all subjectsdid not change significantly as a result of training (Table 4).There were no significant differences in age, height, bodymass, percent body fat, or FFM at baseline or in response totraining among the three IGF1 promoter genotype groups(Table 5).

1 RM strength. The 1 RM strength of the trained leg did notdiffer significantly at baseline among IGF1 promoter genotypegroups when covaried for sex and age. The 192 carriersincreased their 1 RM strength (67.3 6.3 N) significantlymore than noncarriers (39.8 7.6 N) when covaried for sexand age (P 0.05; Table 6; Fig. 1), but all groups increasedsignificantly with training (P 0.01). There was no significantdifference between 192 homozygotes and nonhomozygotes forchange in 1 RM. In response to training, SNP analysis of

Table 2. IGF1 CA promoter allele and genotype frequency

Number of CA Repeats Total (%) Men Women

17 1 (1) 0 117 3 (2) 2 118 8 (6) 5 319 82 (62) 38 4420 27 (20) 12 1521 12 (9) 6 622 1 (1) 1 0Range 622 1722 621

Genotype

19/19 (192/192) 24 (36) 10 1419/ (192/) 34 (51) 18 16/ (Noncarrier) 9 (13) 4 5

CA, cytosine adenine.

Table 3. IGF1 SNP allele and genotype distributionof all subjects

Allele Frequency Genotype Frequency

T C A G TT TC CC AA AG GG

19,779 0.78 0.22 0.59 0.37 0.0440,395 0.79 0.21 0.61 0.35 0.04

82,686 0.39 0.61 0.19 0.40 0.40

Table 4. Physical characteristics for all subjects

Men (n 32) Women (n 35)

Baseline Afte r tra ining Ba seline After train ing

Age, yr 70 (6) 67 (8) Height, cm 169 (6) 161 (8) Weight, kg 83.5 (12.0) 84.0 (12.7) 71.3 (13.6) 71.2 (14.13)Fat, % 29 (5) 29 (4) 39 (5) 39 (6)FFM, kg 58.2 (6) 58.5 (7.1) 42.3 (6.1) 42.3 (5.1)1 RM, N 289 (55) 357 (88) 159 (28) 203 (41)

Values are means with SD in parentheses. FFM, fat-free mass; 1 RM,1-repetition maximum.




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covariance main effects and two-factor interactions revealed nosignificant differences in the change of 1 RM strength.

MV and MQ. MV increased (i.e., increases in trained legminus changes in untrained leg) in all groups (192 homozy-gotes, heterozygotes, and noncarriers) in response to training(P 0.01). MV and MQ did not differ significantly at baselineor in response to training among IGF1 promoter genotype

groups when covaried for sex and age (Table 6; Fig. 2).However, training-induced increases in MV for 192 carriers(132 12 ml) approached significance for being greater thanthe increases in noncarriers (94 18 ml; P 0.08). The 192homozygotes were not significantly different from the het-erozygotes and noncarriers in MV or MQ response to training.Moreover, there were no significant differences in MV re-sponses to training among any SNP main-effect and two-factorinteractions.

DISCUSSION

To our knowledge, this is the first study to examine theinfluence of IGF1 genotype on strength and MV responses toST. These results support our hypothesis that IGF1 genotypeinfluences the strength response to ST but do not support ourhypothesis of a similar influence on MV and MQ responses toST in healthy older adults. However, the genotype associatedwith MV response to training approached significance.

ST appears to be the intervention of choice for the preventionand treatment of sarcopenia, based on efficacy and safety concernswith other interventions (5, 23). IGF-I has been implicated in theloss of muscle with age (23), and IGF-I expression levels changeas a consequence of ST in older adults (16). The results of thisstudy add to this literature by showing that, in older adults, carriers

of the 192 allele at the IGF1 locus have greater strength gains thannoncarriers with ST.

Although no previous studies have examined the associationof the IGF1 gene promoter polymorphism with skeletal musclephenotypes, IGF1 transgenic expression and exogenously ad-ministered IGF-I protein have been shown to increase skeletalmuscle size and function (8, 37). Additionally, muscle stimu-

lation (mechanical, electrical, and stretch) leads to increases inIGF-I protein and overall protein content in skeletal muscle andproduces significant increases in muscle size and strength (15,16, 34, 60). Although a causal relationship between acute orchronic muscle contraction and IGF1 expression has not yetbeen documented in humans, IGF1 remains one of the stron-gest candidate genes for ST-induced increases in muscle massand strength. The increase in IGF1 protein has been reported tobe proportional to the increase in muscle strength in elderlyadults after ST (16). Thus it seems that increases in IGF1expression may be causally related to increases in muscle sizeand strength. In the study by Fiatarone Singh et al. (16), IGF-Iprotein increased by an average of500% in response to STin the elderly. However, the variation in IGF1 mRNA expres-

sion was 500%, suggesting a substantial interindividual vari-ability in response to training.

Indeed, IGF-I blood levels, muscle size, and function areknown to be significantly influenced by genes (9, 24, 28, 50, 55).IGF-I blood levels are highly heritable in children, middle-aged,and elderly adults (24, 28). In this regard, a study examining thesame CA repeat IGF1 promoter polymorphism as the one in thepresent study demonstrated a significant age-related decline incirculating IGF-I levels that was associated with this polymor-phism (44). Consistent with our findings, this would suggest an

Table 5. Physical characteristics for subjects by IGF1 promoter genotype

192 Homozygotes 192 Heterozygotes All Other Genotypes

Baseline After training Baseline After training Baseline After training

Age, yr 671 701 684Height, cm 1692 1672 1653Weight, kg 78.62.5 78.72.5 75.72.0 76.32.1 75.85.3 76.35.6

Fat, % 341 341 341 341 332 341FFM, kg 51.81.4 52.01.4 50.01.0 50.41.0 50.02.2 50.12.4Male/female, n 10/14 18/16 4/5

Values are means SE. None of the after-training values were significantly different from baseline. Weight, %Fat, and FFM are corrected for mean age (68yr) and sex (50/50 ratio).

Table 6. Muscle volume, 1 RM muscle strength, and muscle quality measurements before andafter training by IGF1 192 CA repeat polymorphism

192 Homozygotes 192 Heterozygotes All Other Genotypes

Baseline After training Baseline After training Baseline After training

Trained leg

Muscle volume, ml 1,47033 1,59037* 1,36038 1,51043* 1,33074 1,44075*Muscle quality, N/ml (103) 1576 1875* 1635 1914* 1607 1766*1 RM, N 2318 29812* 2228 2898* 21315 25315*

Untrained leg

Muscle volume, ml 1,41548 1,41348 1,30640 1,31646 1,32276 1,33379Muscle quality, N/ml (103) 1538 1709 1635 1766 13710 15391 RM, N 21712 24011 21311 23111 18118 20419

Values are means SE. *Significantly different from baseline (P 0.01). All values are corrected for mean age (68 yr) and sex (50/50 ratio).




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influence of the IGF1 promoter polymorphism on phenotypes thatmay be affected by IGF1 expression.

The hypothesis that genetic factors may influence muscularstrength is supported by data from both animals (6) andhumans (43, 54, 55). Support from human studies come fromReed et al. (43) who observed significant genetic effects forabsolute grip strength and adjusted grip strength; it was esti-mated that genetic factors accounted for 65% of the variance ingrip strength, even after adjusting for the effects of weight,height, and age (43). Other studies have demonstrated between

31 and 82% of variance in strength is explained by geneticfactors. (54, 55)To our knowledge, only two studies have attempted a genome-

wide search for genes related to phenotypic measures of muscle(10, 11). In one of these studies, FFM, measured by hydrostaticweighing, showed significant linkage with the IGF1 receptorpolymorphism in the Quebec Family Study (10). In the HERI-TAGE Family Study, baseline FFM and the change in FFM dueto exercise training both showed evidence of significant linkagewith the CA repeat IGF1 promoter polymorphism examined inour present study (11). Additionally, linkage and association ofbaseline FFM and the change in FFM due to exercise training hasbeen demonstrated with the IGF1 promoter polymorphism (52).Although the training modality used in the HERITAGE study is

not usually associated with large changes in FFM, it appears thata polymorphism in the IGF1 gene may be associated with changesin FFM. Likewise, we have demonstrated an association of thispolymorphism with muscle function (strength) response to train-ing, which is believed to be under the autocrine/paracrine influ-ence of IGF1.

The IGF1 promoter polymorphism has been examined in nu-merous contexts (20, 29, 44, 46). As in the present study, geno-typing of the microsatellite is typically separated into threegroups: 192 homozygotes, 192 heterozygotes, and noncarriers ofthe 192 polymorphism. Whether the 192 polymorphism is caus-ally related to changes in IGF1 function is not known; yet, the 192allele is the most prevalent allele in the majority of the populations

studied to date. Additionally, there is evidence in animals andhumans that CA repeats found in gene promoter regions can affectgene expression (2, 48). It has been suggested by King et al. (29a)that these repeats act as evolutionary tuning knobs to fine-tunegene expression with minor deleterious consequences. Further-more, genome-wide scans and linkage studies have implicated thisallele in phenotypes known to be affected by IGF-I (11, 14, 52).

As mentioned, the functionality of this polymorphism is notcurrently known. It is possible that length of the polymorphismcould be affecting expression levels or that this polymorphismis simply a marker for phenotypes related to IGF1 expression.Based on the genome-wide scans that have been reportedassociating this polymorphism with IGF1-related phenotypes,it appears that it is at least a marker for IGF1-related pheno-types. To address the functionality of this polymorphism, IGF1expression levels would be needed along with a method ofgrouping that could account for heterozygotes who carried onecopy longer and one copy shorter than 19 repeats. Therefore, inlight of the current lack of data on functionality, the conven-tional grouping of 192 carriers and noncarriers was used aspreviously reported in the literature.

Rosen et al. (46) first implicated this polymorphism withserum levels of IGF-I and bone mineral density in a studyexamining older men and women (46). It was reported that 192homozygotes had lower blood levels of IGF-I; additionally,and in a group of older men, 192 homozygotes made up adisproportionately large number of those with idiopathic os-teoporosis (46). The reports by Frayling et al. (20) and Rietveldet al. (44) concur with Rosen et al.s (46) findings of decreasedblood levels of IGF-I in carriers of the 192 allele. In contrast toRosen et al.s findings, subsequent studies have demonstratedthat 192 noncarriers have lower IGF-I blood levels and aremore likely to be osteoporotic (29, 45). Additionally, a longi-tudinal study by Missmer et al. (36) in middle-aged and older

men and women reported a lower level of IGF-I in controlsubjects who were noncarriers of 192. Some reports haveconcluded that the 192 allele has no effect on blood levels ofIGF-I or only has an effect when combined with oral contra-

Fig. 2. The difference between the change in muscle volume of the trained legand control leg for each IGF1 promoter genotype group corrected for age andsex after 10 wk of training. Carriers of the 192 allele did not differ significantlyfrom noncarriers in terms of their gain in muscle volume ( P 0.08). Valuesare means SE.

Fig. 1. The absolute change in 1-repetition maximum (1 RM) strength cor-rected for age, sex, and baseline muscle strength for each IGF1 promotergenotype group after 10 wk of training. Carriers of the 192 allele hadsignificantly greater gains in strength than noncarriers (*P 0.02). Values are

means SE.




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ceptive use (3, 13, 27, 61). Although conclusive determinationof the effect of the 192 genotype from the aforementionedstudies is difficult, it seems that the IGF1 192 allele is at leastassociated with some phenotypes related to IGF1 expression.Skeletal muscle is known to significantly increase IGF1 ex-pression levels and produce unique isoforms of IGF-I inresponse to ST (16, 34). Yet, none of the previous studies has

examined phenotypes related to skeletal muscle. In the presentstudy, we report the first intervention study to examine theinfluence of this polymorphism on muscle phenotypes knownto be related to IGF1 expression.

Some limitations of the present study are the lack of IGF1expression data for muscle and IGF1 cellular mediators, theuse of only one ethnic group, and the statistical power for MV.The use of one ethnic group was decided on to control for thepossible varying effects this polymorphism might have indifferent ethnic (genetic) backgrounds and because it has beenshown that the prevalence of the 192 allele differs with eth-nicity (13). We are currently examining this relationship inAfrican Americans. The inconclusive results in MV of thepresent study may be due to a lack of statistical power. Thestatistical power to detect the genotype difference in MV was,a-priori, 0.60, whereas for strength it was 0.80 (post hoc,0.66 for MV and 0.95 for strength). This is primarily due tothe smaller (compared with strength) effect size seen in MV inresponse to ST. Additionally, the design of the present studymay have reduced MV effect size by the use of an untrainedcontrol leg. The control leg reduced the absolute change seenin response to ST yet allowed for a more precise determinationof the change that could be attributed to ST or genetics byreducing the likelihood that extraneous factors would affect ourdependent variable of MV. To asses IGF1 muscle expressionand cellular mediators, muscle biopsy samples would be re-quired. However, our focus was to first determine whether

IGF1 genotype is related to our muscle phenotypes of interest.Thus, if our results had demonstrated no relationship, havingthe muscle expression and cellular mediator data might not bevery meaningful, or at least difficult to interpret. Yet, theresults of the present study do suggest the need for a largerscale investigation and acquiring muscle samples to study geneexpression and cellular mediators. Thus future studies shouldconsider changes in muscle mass or MV with a larger samplesize and the use of a control leg or group, consider using otherethnic groups, and, where the muscle biopsy technique isavailable, measure mRNA and/or protein levels of IGF-I.

In conclusion, this is the first study to examine the effects of theIGF1 promoter polymorphism on muscle phenotypic responses toST in older adults. The results suggest that, in response to ST,

carriers of the 192 allele will have a greater increase in strengthand possibly MV compared with noncarriers. The results of thepresent study, in combination with future studies, will continue tocontribute to the understanding of the role of gene polymorphismson the responses to exercise training.

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