Experimental Gerontology 115 (2019) 104–113

Contents lists available at ScienceDirect

Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero

The effects of a combined bodyweight-based and elastic bands resistance T

training, with or without protein supplementation, on muscle mass,
signaling and heat shock response in healthy older people
⁎
Mauricio Krausea,b,c, , Domenico Crognalea,c, Karl Cogana, Serena Contarellie, Brendan Egana,d,
⁎⁎
Philip Newsholmef, Giuseppe De Vitoa,c,
a
Institute for Sport & Health, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Ireland
b
Laboratory of Inflammation, Metabolism and Exercise Research (LAPIMEX) and Laboratory of Cellular Physiology, Department of Physiology, Institute of Basic Health
Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
c
Food for Health Ireland, University College Dublin, Ireland
d
School of Health & Human Performance, Dublin City University, Ireland
e
Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy
f
CHIRI Biosciences Research Precinct, School of Biomedical Sciences, Curtin University, Australia

A R T I C LE I N FO A B S T R A C T

Section Editor: Christiaan Leeuwenburgh This investigation sought to determine the effects of twelve weeks of resistance exercise training in addition to
Keywords: protein supplementation on body composition, markers of muscle atrophy/hypertrophy and heat shock response
Resistance exercise training (HSR) in healthy older adults. Thirty-eight healthy sedentary participants (M/F, 18/20; age, 63.5 ± 4.4 y) were
Protein supplementation randomly assigned to four groups: I) PLACEBO: no training, receiving placebo sachets; II) NUTRITION: no
Muscle growth and function training, receiving protein supplementation sachets; III) EXERCISE PLACEBO: training, placebo sachets and IV)
Heat shock response EXERCISE NUTRITION: training, receiving protein sachets. The resistance training (using bodyweight and elastic
bands) consisted of 45 min supervised training sessions, 3×/week. Participants from both exercise groups in-
creased their total lean body mass (from 48.4 ± 8.7 to 49.2 ± 8.7 kg and from 44.9 ± 7.8 to 45.9 ± 8.1 kg,
average of gain ~0.8 and 1 kg, placebo and nutrition respectively) and improved results in physical tests.
Exercise nutrition group also reduced their body fat (from 34.8 ± 7.3 to 32.9 ± 7.4%), increased the expression
of proteins/gene involved on the HSR, S6 and eEF2, while FOXO3 and Murf1 were reduced. Expression of MHC-I
was reduced in both exercise groups while MHC-IIa increased, with no effect of protein supplementation alone.
Body-weight and elastic bands based resistance exercise prompted, in healthy older people, improvements in
body composition and muscle function. When protein supplementation was added to the people engaged in
resistance training, improvements in fat mass and changes in skeletal muscle signaling were detected, favoring
protein synthesis pathways and the protective heat shock response.

1. Introduction These age-related losses, affecting physical function, can be further

According to the most recent projections in 2030 the number of
older citizens (80+ years) will represent up to 30% of the industrialised
countries population and 12% of the under development ones
(Knickman and Snell, 2002). Aging unfortunately, is associated with a
generalized deterioration of physiological function including a pro-
gressive decline in skeletal muscle mass and muscular strength and
power which progressively translate into functional impairment, in-
creased rate of disability and dependency (Naseeb and Volpe, 2017).
exacerbated by reduced physical activity levels and a suboptimal nu-
tritional profile (Naseeb and Volpe, 2017).
In this context, to preserve muscle function is crucial in aging for
maintaining mobility so reducing the economic and social cost asso-
ciated with possible loss of independence. It is now unanimously re-
cognized that the practice of regular physical exercise is an effective
non-pharmacological therapeutic and preventive intervention delaying
or attenuating the negative effects that aging operates on functional and
metabolic parameters (Leite et al., 2015).

⁎
Correspondence to: M. Krause, Department of Physiology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
⁎⁎
Correspondence to: G. De Vito, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Belfield, Dublin 4, Ireland.
E-mail addresses: mauricio.krause@ufrgs.br (M. Krause), giuseppe.devito@ucd.ie (G. De Vito).

https://doi.org/10.1016/j.exger.2018.12.004
Received 4 September 2018; Received in revised form 28 November 2018; Accepted 4 December 2018
Available online 05 December 2018
0531-5565/ © 2018 Elsevier Inc. All rights reserved.
M. Krause et al.
Table 1
General group characteristics.
PLACEBO NUTRITION
(5♂/5♀) (4♂/3♀)
Age (y) 63.8 ± 4.5 62.3 ± 5.3
Body mass (kg) 73.3 ± 11.5 78.4 ± 13.4
Height (m) 1.66 ± 0.09 1.69 ± 0.1
BMI (kg·m−2) 26.3 ± 2.5 27.2 ± 3.3
Resistance exercise (RE), represents a well-established counter-
measure to lessen the decline in lean body mass (LBM) and muscle
function that occurs with aging (Cruz-Jentoft et al., 2010; Krause et al.,
2015a). However, stable-isotope-based methodologies have demon-
strated that muscle protein synthesis (MPS) in older adults, in response
to exercise or protein supplementation, appear to be reduced or de-
layed, indicating anabolic resistance (AR) (Burd et al., 2013;
Drummond et al., 2008). Despite the presence of AR, present evidence
suggests that protein supplementation may be able to overcome these
issues, particularly when combined with RE programmes (Murton,
2015).
Muscle protein synthesis is stimulated by RE and the time course
and the overall MPS response to this form of exercise have been ex-
tensively investigated (Burd et al., 2013), but the effects of RE training,
on markers of muscle atrophy and heat shock response (HSR) in older
adults is at present under reported. The HSR involves the expression of
heat shock proteins (particularly HSP70) in response to non-lethal
stress to the cells (Krause et al., 2015b). The 72 kDa member of the
70 kDa family of heat shock proteins, HSP70 (encoded by the HSPA1A
gene in humans) is the most abundant of all HSPs accounting for 1–2%
of cellular protein, and is plentiful in skeletal muscle (Noble et al.,
2008). As molecular chaperones, the intracellular HSP70 proteins can
interact with other proteins (unfolded, in non-native state and/or stress-
denatured conformations) to avoid inappropriate interactions, forma-
tion of protein aggregates and degradation of damaged proteins, as well
as helping the correct refolding of nascent proteins (Krause et al.,
2015b). Other functions include protein translocation (Chirico et al.,
1988), anti-apoptosis (Garrido et al., 2001) and anti-inflammatory re-
sponses (Krause et al., 2015c), control of cell signaling (Calderwood
et al., 2007), modulation of immune response (Johnson and Fleshner,
2006), and modulation of chronic disease such as diabetes, obesity and
insulin resistance (Chung et al., 2008).
Recently, evidences for a crosstalk between skeletal muscle protein
synthesis, degradation and HSR signaling has been described (Senf,
2013; Wiggs, 2016). However, HSR may be blunted in older in-
dividuals, but can be potentially restored by RE (de Lemos Muller et al.,
2018). Thus, the aim of the present study was to investigate the effects
of twelve weeks of bodyweight and elastic bands based resistance ex-
ercise training, combined or not with protein supplementation, on body
composition, physical function, markers of muscle atrophy/hyper-
trophy and HSR in healthy older adults.
2. Materials and methods
2.1. Participants
A total of 38 sedentary non-smoking participants (M/F, 18/20; age,
63.5 ± 4.4 y; body mass, 74.3 ± 10.9 kg; BMI, 25.5 ± 2.7 kg·m−2)
volunteered for the present study. Participants were recruited using a
wide variety of techniques, including newspaper advertisements, pos-
ters, distribution of information flyers, and by word of mouth. Written
informed consent was obtained from all participants prior to com-
mencing the study after written and verbal explanation of the study
design approved by the University College Dublin Research Ethics
Committee (RCT number LS-11-212-DeVito). The medical history of the
participants was evaluated, and all were defined as medically-stable
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Experimental Gerontology 115 (2019) 104–113
EXERCISE + PLACEBO EXERCISE + NUTRITION
(5♂/5♀) (4♂/7♀)
63.9 ± 3.9 63.9 ± 4.3
73.6 ± 12.5 71.5 ± 6.1
1.71 ± 0.07 1.68 ± 0.08
25 ± 2.8 25.4 ± 2.3
(Greig et al., 1994). Participants were excluded if they reported a his-
tory of myocardial infarction, cardiac illness, vascular disease, un-
controlled metabolic disease, stroke, sarcopenia [according to the
European Working Group on Sarcopenia in Older People (EWGSOP)
criteria (Cruz-Jentoft et al., 2010)], hormonal replacement, major sys-
temic disease or any condition that would prevent them from engaging
in an exercise study; or if they were already engaging in two or more
planned and structured exercise sessions per week. All subjects were
reported as non-diabetic after interview with an experienced physician
who considered their fasting glycaemia as the main criteria for diabetes
exclusion.
2.2. Study design
This study was a randomized, single blinded, controlled trial of
bodyweight-based and elastic bands resistance exercise training, com-
bined with protein supplementation, performed for 12 weeks (for de-
tails please consult flow chart of patients at the Supplemental material).
Participants were randomly assigned to four groups (Table 1): I) PLA-
CEBO: no exercise training, receiving placebo sachets; II) NUTRITION:
no exercise training, receiving protein supplementation sachets; III)
EXERCISE PLACEBO: exercise training, receiving placebo sachets and
IV) EXERCISE NUTRITION: exercise training, receiving protein sup-
plementation sachets. After completion of baseline assessments, the
randomization procedure was stratified by gender, based on equal
distribution of males and females, but randomized to respective inter-
vention groups thereafter, without blocking. The control group was
instructed to maintain their usual physical activity and dietary habits
for the duration of the study. The exercise training intervention con-
sisted of 45 min supervised group training sessions performed three
times per week for 12 weeks, with each session consisting of body-
weight-based resistance exercise and elastic band exercises. Assess-
ments of body composition and physical function took place at baseline
(PRE) approximately one week before commencing the intervention,
and after 12 weeks of intervention (POST). This study was part of a
previous joint project between two research institutions were the pri-
mary outcome variable was change in lean body mass (Francis et al.,
2016). The estimated sample size was calculated for a larger study,
using the data from Tieland et al. (2012), based on an increase in LBM
of 1.1 ± 1.4 kg (mean ± SD). A minimum of 20 participants per
group was required to detect a difference (α = 0.05 and β = 0.8). In the
present study, however, we analyzed a different sub-group of people
(no additional sample size calculation was performed; n = 38, ~10 per
group;) where skeletal muscle biopsy samples were obtained to eval-
uate markers of gene/protein expression at PRE and POST, looking at
genes and proteins involved on protein synthesis/degradation, meta-
bolism, inflammation and heat shock response.
2.3. Bodyweight-based and elastic bands resistance training intervention
Supervised (by qualified sport and exercise scientists) exercise was
performed in a gym on three non-consecutive days of the week. Each
session lasted 45–60 min and consisted of a warm up, the bodyweight-
based resistance exercise training and a cool down. Compliance to the
exercise program was monitored using a weekly exercise log which was
checked by the trainers administering the exercise session. The first
M. Krause et al.
3 weeks of the program had an emphasis on ensuring the correct ex-
ercise technique and monitoring, for everyone, the appropriate amount
of exercise and rest intervals. Between weeks 3 and 12, the program
was designed to promote muscle hypertrophy (4–6 sets, 8–15 repeti-
tions) as recommended by Bird et al. (Bird et al., 2005). The training
consisted of a combination of upper and lower body exercises using
thera-bands (Therabands resistance loop, USA) and body weight as the
primary resistance. The primary exercises used throughout the program
included squats, lunges, hip abduction, shoulder press, latissimus dorsi
pull-down, bicep curls, calf raises, push-ups, triceps dips and lumbo-
pelvic stabilisation exercises. Thera-band exercises were progressed by
increasing the resistance of the band in ascending order: red, green,
blue and black respectively. Progression was determined based on the
participants post exercise rate of perceived exertion (RPE) in con-
sultation with the exercise trainer. Participants received at least 30 s
rest between sets. The training program was set out in 4 × 3 week
progressive cycles (Cycle 1: 3 sets of 8–12 reps; Cycle 2: 3 sets of 14
reps; Cycle 3: 4 sets of 10–12 reps; Cycle 4: 4 sets of 12–14 reps). A full
description of the training program can be found elsewhere (Francis
et al., 2016).
2.4. Protein supplementation and placebo
Participants were instructed to take a supplement at their two lower
protein containing meals of the day (typically breakfast and lunch).
Sachets were provided in powder format and could be mixed with water
to make-up a powdered beverage. Supplements were prescribed relative
to the median 4 levels of participants' body mass (BM) (i.e. 45–59.9 kg,
median of 52.5 kg; 60–74.9 kg, median of 67.5 kg; 75–89.9 kg, median
of 82.5 kg and 90–105 kg, median of 97.5 kg). Each protein supplement
dose provided 0.165 g protein kg−1 BM day−1 of median BM. Five fla-
vors were available to off-set flavor fatigue and to improve compliance.
All used or unused sachets were returned by the subjects for intake
control. Subjects were blinded to the supplement composition assigned
to them. Compliance was monitored by count-back at the end of each
month. Placebo consisted of an isoenergetic, non-nitrogenous mal-
todextrin sachet that was ingested at breakfast and midday meals.
The milk protein matrix was composed of a 9:2:1 ratio of milk
protein concentrate [80% (wt:wt protein); Glanbia Ingredients Ireland,
Kilkenny Ireland]; whey protein concentrate hydrolysate, degree of
hydrolysis 32% [WPC DH 32. 78% (wt:wt protein); Carbery Food
Ingredients, Cork, Ireland; and whey protein isolate hydrolysate, degree
of hydrolysis 45% [WPC DH 45, 75% (wt:wt protein); Glanbia
Ingredients Ireland]. The total protein concentration was 72.7 g/100 g
powder. The protein matrix was supplemented with 2187 mg/100 g
powder of milk-based calcium (Trucal; Glanbia Ingredients Ireland) and
57.3 mg/100 g powder cholecalciferol. A full breakdown of the sup-
plement composition has recently been published (please consult
(Norton et al., 2016) and Supplementary Table 1).
2.5. Anthropometry and body composition
Standing height was measured using a Stainless Steel Harpenden
Stadiometer (Holtain Ltd, Pembrokeshire, United Kingdom), with the
participants' shoes off and head at the Frankfort horizontal plane. Body
mass was measured using a Seca 888 weighing scale, Birmingham,
United Kingdom. Body composition was assessed in the overnight
fasted state by dual energy X-ray absorptiometry (DEXA) (Lunar iDXA,
GE Healthcare, Buckinghamshire, United Kingdom). DXA quality as-
surance measurements and calibrations were performed daily to ensure
scanner reliability, and identical patient scan protocols were performed
for all participants.
2.6. Assessments of physical function
The ability to rise from a chair was assessed using a timed 5
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Experimental Gerontology 115 (2019) 104–113
repetition chair rise test and by counting the number of chair rises
completed in 30 s (Forte et al., 2013). Lower body flexibility was as-
sessed using the chair version of the sit & reach test (Forte et al., 2013).
Functional ability assessment included walking speed measurement
performed on a 10 m indoor course using measuring gates (Smartspeed,
Fusion Sport, Coopers Plains, Australia). Participants were asked to
walk “as fast as possible without running” for maximal walking speeds
assessment. Isometric hand grip strength of both hands was measured
after adjustment for hand size, with the participant standing and re-
laxing their arm along their body (Baseline Hydraulic Hand Dynam-
ometer Fabrication Enterprise Inc. Irvington NY). It is advisable to
measure hand grip in both arms, since significant differences between
the dominant and the non-dominant side have been observed for
handgrip strength, in both sexes (Ditroilo et al., 2010). Two trials were
performed with 1 min rest in between and the highest score used for
statistical analysis. All measurements were carried out by the same
exercise scientist to exclude issues with inter-tester reliability.
2.7. Blood pressure and biochemistry
Systemic arterial blood pressure (BP) was measured in the brachial
artery using an Omron M5-1 fully automated BP monitor (HEM 907,
Omron Healthcare, Kyoto, Japan). The mean of two measurements
taken at 2 minute intervals of supine rest was recorded. Venous blood
samples were taken after fasting from an antecubital vein in heparin
coated and gel-clot vacutainerTM tubes using standard aseptic techni-
ques. Samples were immediately centrifuged (at 4 °C and 1000 g for
15 min), after which plasma and serum was removed and stored at
−80 °C for further analysis.
2.8. Plasma IGF-1 and IGFBP3 quantification
A highly sensitive, enzyme-linked immunosorbent assay method
was used to determine the levels of IGF-1 and IGFBP3 (Quantikine
ELISA Kit, R&D Systems, USA) in plasma samples. Absorbance was
measured at 450 nm, and a standard curve constructed from known
dilutions of the proteins. Quantification was made using a microplate
reader (Molecular Devices SpectraMax Plus 384, Sunnyvale, California,
United States). The intra- and inter-assay CV, respectively, ranged be-
tween 4.3 and 5% for IGF-1 and 3.2 and 7% for IGFBP3.
2.9. Muscle biopsy
Skeletal muscle biopsies were obtained from the vastus lateralis
muscle before (PRE) and after (POST) the 12-week intervention. The
POST biopsy was taken between 48 and 72 h after the final training
session. Muscle biopsies were obtained from separate sites (2 cm apart)
from the lateral portion of the muscle. Biopsies were obtained using a
semi-automatic spring-loaded biopsy system (MEDAX/BF14100-C0;
Bio-Feather with coaxial 14 g 10 cm). Samples were obtained (about
30–50 mg) under local anesthesia (0.2% lidocaine) and immediately
frozen in liquid nitrogen and stored at −80 °C for later analysis.
2.10. Protein quantification and Western blot
Briefly, the tissues were homogenized (still frozen) in 0.1% (w/v)
SDS buffer containing protease and phosphatase inhibitor cocktail
(Sigma). Afterwards, the homogenates were centrifuged at 15000 ×g
for 5 min at room temperature and the supernatant fractions were saved
for protein determination by using bovine serum albumin (BSA, Sigma)
as standard. Then, equivalent amounts of protein from each sample (~
40 μg) were mixed with Laemmli's gel loading buffer in a ratio of 1:1,
boiled for 5 min and electrophoresed. The samples were separated by
SDS-PAGE (4–15% MiniPROTEAN TGX™ Precast Protein Gels, Bio-Rad)
and transferred onto nitrocellulose membranes (GE Healthcare Life
Sciences “Amersham Protran 0.2 NC”). Non-specific binding was
M. Krause et al.
blocked in a 7.5% milk/2.5% BSA/TBS-t (10 mM Tris pH 7.5, 100 mM
NaCl, 0.4% Tween20) for 1 h at room temperature. Membranes were
incubated overnight with primary antibodies directed towards the fol-
lowing primary antibodies: eEF2 (1:1000; Cell Signaling Technology,
Beverly, MA; no. 2332), p70 S6 Kinase (1:1000; Cell Signaling; no.
9202), S6 Ribosomal Protein (5G10) (1:1000; Cell Signaling; no. 2217),
HSF1 (1:750; Cell Signaling; no. 4356), GAPDH (1:7500; Cell Signaling;
no. 2118), HSP70 (1:2000; Sigma-Aldrich (St. Louis,MO, USA; no.
H5147). Membranes were washed in TBS-t and incubated with appro-
priate secondary HRP-conjugated antibodies (Bio-Rad), visualized by
ECL (Pierce™ ECL, Thermo Fisher Scientific) and quantified on ImageJ
software. GAPDH were used for normalization.
2.11. Quantitative polymerase chain reaction (qPCR) analysis of gene
expression
Total RNA was isolated from approximately 20 mg of crude muscle
using TRIzol reagent (Sigma-Aldrich, UK) as per the manufacturer's
instructions. Total RNA concentration was quantified spectro-
photometrically at an absorbance of 260 nm (NanoDrop ND-1000
Spectrophotometer, ThermoFisher Scientific, Waltham, MA, USA). The
integrity and purity of each RNA sample was verified by measuring the
spectrophotometric A260/A280 (> 1.8) and A260/A230 (> 1.5) ra-
tios. RNA (1 μg) was reverse transcribed to cDNA using the High
Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as per
the manufacturer's instructions. The cDNA template was stored at room
temperature until subsequent analysis. Relative mRNA expression
(20 ng cDNA template per reaction) was determined using quantitative
real-time PCR (ABI Prism 7900HT, Applied Biosystems, Foster City, CA,
USA) using Assay-On-Demand primer pairs and probes (P/N 4331182,
TaqmanH Gene Expression Assays, Applied Biosystems) and TaqmanH
Universal PCR MasterMix (Applied Biosystems). Assay IDs for specific
mRNA targets are listed in Table S1. GAPDH mRNA expression
(4333764F, Applied Biosystems) was stable across time points during
the training intervention and used as the housekeeping gene to which
target mRNA expression was normalized.
2.12. Statistical analysis
The GraphPad Prism (v6.0; IBM Corp.) statistics and graphical
software package was used for all analyses. Descriptive data are re-
ported as mean ± SD. Two-way (time x group) repeated measures
analysis of variance (ANOVA) was used to detect differences, with
pairwise comparisons performed using Bonferroni post-hoc test when a
significant main or interaction effect was detected. For all analyses,
statistical significance was accepted at p < 0.05.
3. Results
3.1. Body composition
Table 1 displays baseline participants' characteristics. There were no
differences at baseline between groups in terms of age, BMI, body mass
and height. After 12 weeks of intervention, participants from both ex-
ercise groups, protein supplementation or placebo (p = 0.001 and
p = 0.0058; Cohen's D effect size: 0.26 and 0.08, respectively), increased
their total lean body mass in a similar manner while the 2 non-exercise
groups did not change (Fig. 1A). No differences were found for total
lean mass gain between the exercise groups (nutrition vs. placebo),
however, only in the exercise nutrition group a significant reduction in %
of body fat was observed (p < 0.0001; Cohen's D effect size: 0.139)
(Fig. 1B). No changes were found in bone mineral density (Table 2).
3.2. Functional physical tests
In both exercise groups the time taken to complete 5 chair rises was
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Experimental Gerontology 115 (2019) 104–113
reduced whereas the number of chair rises completed in 30 s increased
(Cohen's D effect size: 0.58 and 0.32, respectively, Fig. 1D and E). No
changes were found in the 10 m walk test (Fig. 1F), however, both
exercised groups increased their flexibility, based on the sit and reach
test results (Cohen's D effect size: 0.355 and 0.05, respectively, Fig. 1G).
As expected, considering that the training was not specific for upper
body, no changes were detected for handgrip strength (Fig. 1C).
3.3. General blood biochemistry
No changes were found for any of the considered parameters
(Table 2).
3.4. Gene and protein expression
Skeletal muscle samples were taken after 48–72 h to minimize any
residual effect of the last training session. After 12 weeks of interven-
tion exercise nutrition group increased the levels of HSF-1 (p < 0.0001),
S6 (p < 0.05), HSP72 (p < 0.05) and eEF2 (p < 0.01). No changes
on P70 levels were found (Fig. 2). FOXO3 (p < 0.05) and Murf1 were
reduced only in exercise nutrition group and no changes on HSP72 or
MAFbx mRNA levels were observed (Fig. 3A, B, C and D). Interestingly,
levels of MHC-I reduced (p < 0.01 for exercise nutrition group and
p < 0.05 for exercise placebo group) and MHC-IIa increased in both
exercise groups after the intervention (p = 0.019 for exercise nutrition
group and p < 0.01 for exercise placebo group) without any additional
effect of protein supplementation (Fig. 3F, G and H). Exercise nutrition
group also reduced MHC-IIx and MyoD expression after intervention
(p < 0.01). No significant differences were found for gene expression
of KLF15, PGC-1α, IL1-β, CCL2, IGF1 or MSTN (data not shown).
4. Discussion
4.1. Bodyweight-based resistance exercise training increases muscle mass
and function in healthy older people, independently of protein
supplementation
Progressive resistance training (PRT) has been clearly shown ef-
fective for both strength and muscle mass enhancement in older in-
dividuals and has been widely used in this population. However, PRT
may not represent the first choice of exercise for the older, especially
women, who may prefer other kind of activities (Cadore et al., 2014;
Rodrigues-Krause et al., 2016). In the present work, it has been shown
that a bodyweight-based resistance exercise intervention [inexpensive
and with a good adherence (compliance of the exercise groups ~85%)],
can induce, in healthy older people, improvements in body composition
(mainly lean mass) and muscle function. When protein supplementa-
tion was added to the diet of the people engaged on the resistance
training, additional gains could be detected, including lower fat mass
and changes in skeletal muscle signaling (protein synthesis pathways
and heat shock response).
These results are even more important because were obtained in
relatively young older individuals and with a proper information and
access to healthy food, especially in respect to the protein intake. So far,
evidence from several studies shows no clear benefit of protein sup-
plementation on skeletal muscle mass in older people, since some stu-
dies found positive effects (Bonnefoy et al., 2003; Borsheim et al., 2008)
and others did not (Candow and Chilibeck, 2008; Verdijk et al., 2009;
Verhoeven et al., 2009). Despite that, long-term interventions have
shown that protein supplementation improves physical performance,
but does not necessarily increase skeletal muscle mass in frail older
people (Tieland et al., 2012), indicating that protein supplementation
(along with exercise) may induce improvements in neuromuscular
control and skeletal muscle only when combined with an appropriate
exercise training. Interestingly, in our twelve-week intervention, the
increased anabolic signaling, induced only in people engaged on
M. Krause et al.
Fig. 1. Changes in whole-body lean body mass and body fat mass assessed by DXA (A and B). Pre and post results of Tests of physical performance: Handgrip strength
(C), chair test (D and E), 10 meters walk test (F) and sit and reach test (G). Data are mean ± SD. Data for body composition are shown as box-and-whisker plots, and
error bars represent Tukey confidence intervals. *p < 0.05.
exercise and protein supplementation (Exercise Nutrition Group), was
not translated into differences in skeletal muscle mass or in physical
function. In fact, both exercise groups increased their muscle mass and
improve their functional test results. However, these changes in protein
signaling may be important, in long term, for exercise-induced adap-
tations (e.g. skeletal muscle mass and function maintenance, insulin
sensitivity, inflammatory control, etc.) that will be translated in main-
tenance of muscle size and function.
Regarding the functional tests results, it is important, however, to
consider the data with caution, since the Exercise Placebo Group started
from a slightly lower functional capacity (considering the chair test
results).
4.2. Protein supplementation increases skeletal muscle anabolic signaling
only when associated with exercise training
Although early evidence suggests a reduced basal MPS rate (i.e.
following an overnight fast) in the older compared with young subjects
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Experimental Gerontology 115 (2019) 104–113
(Koopman and van Loon, 2009), the majority of studies have found that
the rates of muscle protein turnover are not abnormal in older in-
dividuals under basal conditions (Markofski et al., 2015), indicating
that other factors that influence muscle protein turnover may be re-
sponsible for muscle atrophy during aging. Physical training has been
shown to reduce the age-associated loss of muscle strength and the
increased muscle fat infiltration in elderly people (Goodpaster et al.,
2008). Moreover, MPS can be stimulated by both resistance exercise
and nutrition in older people (Drummond et al., 2008). However, the
MPS response to different exercise stimuli is reduced, indicating ana-
bolic resistance (AR).
Whey proteins are a rich source of essential amino acids and rapidly
elevate plasma amino acids, thus providing the foundations for pre-
servation of muscle mass. Previous studies involving supplementation
with whey protein have been shown to be effective in augmenting the
effects of resistance exercise, particularly when supplementation occurs
in the hours surrounding the exercise training (Hayes and Cribb, 2008).
Not only leucine, but other amino acids may be involved on the
M. Krause et al. Experimental Gerontology 115 (2019) 104–113

Table 2
Body composition and general biochemistry responses.
PLACEBO NUTRITION EXERCISE + PLACEBO EXERCISE + NUTRITION
(5♂/5♀) (4♂/3♀) (5♂/5♀) (4♂/7♀)

PRE POST PRE POST PRE POST PRE POST

Urea (mmol/L) 6.12 ± 0.9 4.78 ± 0.8 5.13 ± 0.64 5.39 ± 1.04 5.86 ± 1.87 4.93 ± 1.61 5.5 ± 1.24 5.82 ± 1.88
Creatinine (μmol/L) 71.70 ± 11.94 74.00 ± 12.35 67.00 ± 13.17 68.25 ± 11.99 75.20 ± 23.18 76.30 ± 21.47 74.09 ± 17.52 72.91 ± 8.85
Albumin (mmol/L) 39.40 ± 1.58 37.50 ± 1.65 35.39 ± 12.78 37.00 ± 1.69 36.12 ± 11.37 36.60 ± 2.12 38.45 ± 3.56 36.80 ± 2.25
Total Cholesterol (mmol/L) 5.11 ± 0.69 4.35 ± 0.45 4.88 ± 0.41 4.78 ± 0.81 5.03 ± 0.72 5.01 ± 0.87 5.03 ± 1.04 4.70 ± 0.96
Tryglicerides (mmol/L) 1.48 ± 1.13 1.08 ± 0.75 1.17 ± 0.63 1.47 ± 0.96 1.00 ± 0.28 1.05 ± 0.37 1.08 ± 0.62 1.07 ± 0.68
HDL (mmol/L) 1.24 ± 0.32 1.18 ± 0.29 1.48 ± 0.40 1.21 ± 0.44 1.27 ± 0.34 1.28 ± 0.41 1.56 ± 0.48 1.42 ± 0.46
LDL (mmol/L) 3.16 ± 0.60 2.32 ± 0.46 2.61 ± 0.70 2.53 ± 0.86 2.94 ± 0.50 2.83 ± 0.56 2.69 ± 0.92 2.37 ± 0.95
Calcium (mmol/L) 2.26 ± 0.09 2.10 ± 0.13 2.32 ± 0.07 2.04 ± 0.06 2.34 ± 0.08 2.08 ± 0.08 2.32 ± 0.15 2.10 ± 0.09
Vitamin D (nmol/L) 74.20 ± 21.16 61.70 ± 21.37 69.25 ± 23.42 63.50 ± 22.56 76.20 ± 17.55 61.40 ± 18.91 68.27 ± 25.02 80.27 ± 17.32
BMD (g/cm2) 1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.2 1.1 ± 0.2 1.1 ± 0.1 1.1 ± 0.1
Total Lean Body Mass (Kg) 46.7 ± 10.1 46.7 ± 10 48.4 ± 11.6 48.9 ± 11.5 48.4 ± 8.7 49.2 ± 8.7 44.9 ± 7.8 45.9 ± 8.1
Body Fat (%) 34.4 ± 6.3 33.9 ± 6.9 36.8 ± 13.17 35.6 ± 8.1 32.8 ± 4.9 31.9 ± 5 34.8 ± 7.3 32.9 ± 7.4
Glucose (mmol/L) 5.54 ± 0.43 5.26 ± 0.31 5.68 ± 0.63 5.4 ± 0.5 5.36 ± 0.35 5.06 ± 0.64 5.4 ± 0.37 5.05 ± 0.88
Insulin (mlU/L) 13.71 ± 8.8 11.09 ± 5.09 10.04 ± 3.28 12.4 ± 11.9 7.63 ± 5.35 10.58 ± 6.48 7.49 ± 4.12 10.41 ± 6.5
HOMA-IR (AU) 3.44 ± 2.45 2.61 ± 1.25 2.57 ± 1 3 ± 3.1 1.87 ± 1.4 2.42 ± 1.6 1.78 ± 0.95 2.41 ± 1.64
IGF-1 (ng/mL) 118.2 ± 28.9 161.2 ± 47.7 155.5 ± 92.5 144.1 ± 45.1 129.7 ± 38.3 145.8 ± 61.3 113.1 ± 44.5 139.1 ± 58.1
IGFBP3 (ng/mL) 71.2 ± 18.9 73.4 ± 21.3 66.7 ± 10.4 76.7 ± 10.7 65.7 ± 13.3 74.34 ± 15.2 72.5 ± 21.4 78.3 ± 3
IGF-1/IGFBP3 1.78 ± 0.6 2.39 ± 1.06 2.33 ± 1.33 1.91 ± 0.63 2.01 ± 0.54 1.98 ± 0.68 1.55 ± 0.3 1.82 ± 0.68

With the except of lean body mass and fat mass (* different from baseline, p < 0.05), no other differences were found.

anabolic effects of whey protein supplementation, such as arginine,
cysteine and glutamine. Although the plasma concentration of amino
acids has not been measured in the present study, based on previous
studies (Farup et al., 2016), it is reasonable to expect that, these amino
acids, especially leucine, arginine, cysteine and glutamine should be
increased after the supplement ingestion (please consult Supplementary
Table 1 which describes the contents of the supplement drink). In fact,
at least when consumed after exercise (recovery period), whey protein
and leucine supplementation, resulted in significant increments on
plasma glycine, arginine, glutamine, and leucine (Nelson et al., 2013).
Our results indicate that protein supplementation can potentiate the
effects of exercise on skeletal muscle signaling, particularly increasing
anabolic and decreasing catabolic pathways. The additional effects of
protein supplementation over this signaling may be explained by the
increased availability of specific amino acids, such as leucine, and di-
peptides, including glutamine dipeptides. In fact, whey protein hydro-
lysate, is a rich source of these components. In the case of leucine, the
manner in which this amino acid elicits these anabolic effects within the
muscle cell appears through stimulation of the mammalian target of
rapamycin complex 1 (mTORc1) signaling cascade (Murton, 2015).
This is achieved by mTORc1 inducing two main effects. Firstly, through
the activation of p70 ribosomal S6 kinase, and secondly, by inactivation
of the eukaryotic initiation factor 4E binding protein 1 (eIF4B), a pro-
tein that is known to repress protein translation. Other key downstream
effectors of mTORC1 signaling are the ribosomal protein S6 (S6) and
eukaryotic elongation factor 2 (eEF2), where the activation of these
proteins will finally lead to protein synthesis. In the present interven-
tion, protein supplementation, to people engaged in the resistance
training, resulted in increased expression of S6 and eEF2, indicating a
signaling favoring protein synthesis that may be stimulated by the in-
creased leucine delivered to the skeletal muscle.
The anabolic signaling for adaptive hypertrophy induced by ex-
ercise can involve the rise in blood anabolic hormones such as growth
hormone (GH) and insulin-like growth factor (IGF)-I, and consequent
activation of a signaling cascade through phosphatidylinositol 3-kinases
(PI3K)-Akt-mTOR by IGF-I (Egan and Zierath, 2013). However, there
were no changes on plasma levels of neither insulin, IGF-1 or IGFBP3,
indicating that local modulators may be involved in the activation of
the studied pathways, thus potentially involving IGF-I-independent
mechanisms of mTOR activation through mechano-sensory regulation
(Philp et al., 2011).
4.3. Resistance training along with protein supplementation increases heat
shock response signaling in skeletal muscle and reduces key catabolic factors
To the best of our knowledge, this is the first published work that
evaluates how exercise (resistance), alone or combined with protein
supplementation, modifies HSP70 expression and HS response in older
individuals. Induction of HSP gene expression is regulated by the in-
teraction between the heat shock transcription factors (mainly HSF1)
and the regulatory heat shock elements (Krause et al., 2015a). Based on
its functions, it is predictable that HSP70 may protect against muscle
atrophy through several different mechanisms (Wiggs, 2016); by its
ability to serve as a chaperone; by acting to maintain mitochondrial
integrity; by prevent disuse atrophy induced-proteolysis (through in-
hibiting of the activation of FOXO3 and NF-kB signaling pathways)
(Senf, 2013; Senf et al., 2010); and finally, by maintaining the rate of
translation elongation of nascent polypeptides (Ku et al., 1995; Nelson
et al., 1992). Thus, strategies to increase HSP70 and HSF-1 may induce
beneficial effects for skeletal muscle development in the older in-
dividuals.
Glutamine (and its precursors, e.g. glutamate) availability, for ex-
ample, can improve cellular heat shock response (HS response - capacity
to increase HSP70 in response to non-lethal stress) by enhancing HSF1
activation and expression in cultured cells and also in skeletal muscle of
trained rats (Leite et al., 2016; Petry et al., 2014; Xue et al., 2012). This
can be done mainly by glutamine role on the activation of the hex-
osamine biosynthetic pathway (HBP), that will results in activation of
HSF-1, enhancing HSP70 expression (Krause et al., 2015b). Thus, the
increased availability of glutamine and/or glutamine dipeptides to
people under the exercise protocol, may have resulted in improvements
of the HS response, explaining our findings with regard to HSF-1 and
HSP70 expression. In this regard, it is important to consider, that our
population was composed by healthy older adults that ingest an average
of 1.0–1.2 g protein/kg/day (Candow et al., 2006; Iglay et al., 2007;
Verdijk et al., 2009), a protein intake that would result in sufficient
glutamine to induce a heat shock response. Moreover, recent evidences
have shown that increments in glutamine availability, combined with
exercise (a mandatory stress), can induce further increments on HSP70
expression through higher HBP flux (Leite et al., 2016).
In the present study, exercise without protein supplementation did
not induce changes in muscle HSP70 content. Despite previous studies
reporting that resistance training increases HSP70 in muscle, it is im-
portant to note that the majority of this previous research was

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M. Krause et al.
Fig. 2. Changes in muscle protein expression by Western blot. A. Representative gels; Expression of B. HSF-1, C. HSP70, D. eEF2, E. P70 and F. S6. Data are
mean ± SD. *p < 0.05.
conducted on active healthy young people (Liu et al., 2000; Paulsen
et al., 2012). Some studies involving older people even showed de-
crease of HSP70 after resistance exercise in lymphocytes (Bautmans
et al., 2005). In addition, in animal models and isolated fibroblasts and
lymphocytes from elderly human donors, it has been demonstrated that
aging may induce declines in the HSR (as measured by their capacity to
express iHSP72) (Blake et al., 1991; Deguchi et al., 1988; Njemini et al.,
2002; Njemini et al., 2004). In aged rats (Blake et al., 1991), for ex-
ample, the expression of HSP72 (in brain, lung and skin tissues), is
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Experimental Gerontology 115 (2019) 104–113
reduced after heat shock. In human studies, the heat shock-induced
HSP72 was blunted in older people in comparison with their younger
counterparts (Njemini et al., 2002; Njemini et al., 2004). Despite that, it
cannot be excluded that a longer exercise intervention may induce in-
crements in HSP70 content even in the absence of any supplementation.
As previously discussed, HSP70 prevent disuse atrophy induced-
proteolysis (through inhibiting of the activation of FOXO3 and NF-kB
signaling pathways) (Senf, 2013; Senf et al., 2010). Another point of
crosstalk between HS response and protein synthesis may occur at the
M. Krause et al. Experimental Gerontology 115 (2019) 104–113

Fig. 3. Changes in transcript abundance for markers of muscle atrophy, regulators of growth, HSP70 and myosin heavy chain isoform. Relative mRNA expression was
determined using qPCR and normalized to the housekeeping gene β-actin. A, HSP70. B, FOXO3. C, MuRF1. D, MAFbx. E, MyoD. F, MHC-I. G, MHC-IIA. H, MHC-IIX.
Data are mean ± SD. *p < 0.05.

level of eIF2 protein (a downstream protein of the mTOR pathway). In
fact, eIF2 promotes the activation of HSF1 (Dokladny et al., 2015),
leading to the expression of HSPs under stress conditions. It has been
recently demonstrated, that HSP70 inhibits FOXO3-dependent tran-
scription in a gene-specific manner. Specifically, Hsp70 inhibited
FOXO3a-induced promoter activation of atrogin-1(MAFbx) (Senf et al.,
2010). In the present work, the group taking the supplementation and
performing exercise, increased their HS response protein expression and
this co-occur with a higher expression of eIF2, lower gene expression of
FOXO3 and lower MAFbx expression. As a result, protein

111
M. Krause et al.
supplementation, only in the trained group, seems to sensitize skeletal
muscle cells to respond to anabolic stimulus through increased HS re-
sponse and protein synthesis signaling.
4.4. Bodyweight-based resistance exercise training induce changes in
skeletal muscle myosin heavy chain isoforms
Our intervention, in both groups of exercise, induced a change in
the skeletal muscle Myosin heavy chain isoforms (MHCs expression).
MHC expression was decreased for type I (both placebo and protein
group) and type IIX (only in the protein group) and increased for type
IIA muscle after the exercise intervention. Similar results were identi-
fied previously, confirming that skeletal muscle plasticity, in response
to resistance training, is still present, even at older ages (Sharman et al.,
2001). It is commonly reported that in aging muscles the reduction in
individual fiber size is mainly confined to fast type II fibers, leading to a
progressive decrease in the type II-to-type I fiber area ratio, indicating a
selective atrophy of type II fibers (Korhonen et al., 2006). Muscle
wasting depends on the activation of two major pathways, the protea-
somal and the autophagic-lysosomal systems, both controlled by
FOXO3. As discussed, other pathways, such as NF-κB, are also involved.
FOXO3 activity is downregulated by several factors such as Akt, HSP70
and the transcriptional co-activator PGC-1α, where the last being more
abundant in slow oxidative than in fast glycolytic muscle fibers. The
fact that our resistance exercise training resulted in, perhaps (at least
concerning the genes expression), a shift of muscle fiber type, from
oxidative type I to more fast type IIA may represent a positive adap-
tation to preserve muscle function, muscle size and functional activity.
However, it is important to analyze the data with caution, since no
histological measurements were performed.
5. Conclusions and perspectives
Herein we have shown that twelve weeks of an inexpensive body-
weight and elastic bands based resistance exercise intervention, was
able to induce, in healthy older persons, improvements in body com-
position and muscle function. When protein supplementation was
added to the diet of the people engaged in the resistance training, ad-
ditional improvements in fat mass (↓) and changes in skeletal muscle
signaling, favoring protein synthesis pathways and the protective heat
shock response were detected.
Conflict of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Food for Health Ireland and Enterprise
Ireland Grant CC/2008/001.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.exger.2018.12.004.
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