Effect of 1-year daily protein supplementation and physical exercise on muscle protein synthesis rate and muscle metabolome in healthy older Danes: a randomized controlled trial

The CALM trial (Counteracting Age-Related Loss of Muscle Mass) was conducted at Bispebjerg Hospital between 2014 and 2018. It was designed as an intention-to-treat randomized controlled study. The study reported in this paper was conducted on a subgroup of subjects, who in addition to the general measurements performed in the CALM trial also participated in an acute 1-day stable isotope tracer infusion trial before and after the intervention. The primary outcome of the study was the comparison of basal overnight fasted muscle protein synthesis rate as well as muscle protein synthesis rate response to protein intake from basal overnight fasted state between baseline and after 12 months. We further explored any sex-specific changes regarding the muscle protein synthesis rates as well as changes in the skeletal muscle metabolome. The exploratory analysis was not pre-specified.

Further information and detailed description of the purpose, methods and exclusion criteria in the CALM trial has been published previously [19]. The trial protocol (H-4-2013-070 and H-4-2013-070.3) was approved by the regional ethics committee and all participants provided written informed consent. The trial protocol for this study was registered at clinicaltrials.gov journal number: NCT02115698.

Participants

This study includes 66 healthy older adults above 65 years of age. All participants were screened by a physician prior to enrollment. Participants were excluded if they performed > 1 h of heavy resistance training per week and if they had any medical condition potentially preventing them from completing the 1-year intervention. Participants were allowed to be medicated against hypertension, hypercholesterolemia and thyroid dysfunction. For an exact list of accepted medications see the trial protocol [19].

Study design

After enrollment, participants were randomized using minimization (software MinimPy 0.3; http://minimpy.sourceforge.net/) and stratified by sex and number of completed repetitions on the 30-s chair stand test (< 16 or ≥ 16) into one of five intervention groups: (1) carbohydrate supplementation (CARB; 20 g maltodextrin + 10 g of sucrose), (2) collagen protein supplementation (COLL; 20 g bovine collagen protein hydrolysate (ATpro 200) + 10 g sucrose), (3) whey protein supplementation (WHEY; 20 g whey protein isolate (LACPRODAN, Arla Foods Ingredients P/S, Viby J, Denmark) + 10 g of sucrose), (4) heavy resistance training with whey protein supplementation (HRTW), (5) light-intensity training with whey protein supplementation (LITW). All groups were instructed to dissolve their respective powder-based supplement in water, juice or milk two times daily at breakfast and lunch. All supplements were developed and packaged by Arla Foods Ingredients Group P/S, Viby J, Denmark (for further information, see Bechshøft et al. [19]). The HRTW group followed a supervised center-based progressive heavy resistance exercise program three times weekly and the LITW group was instructed to do a home-based non-supervised progressive light-load resistance training program three to five times weekly using TheraBand® rubber bands (Hygenic Corp., Akron, OH, USA) and bodyweight. For further details, see prior publication [20]. Before and after the 1-year intervention, all participants went through a thorough battery of tests (see previous publication [19]) and an acute stable isotope tracer infusion trial. The present paper primarily reports results from these acute trials (see below).

Acute trial

Participants arrived at the facility 8 a.m. in the morning by car or public transportation to avoid physical activity in an overnight fasted state from 9 p.m. the day before. They were instructed to abstain from strenuous physical activity 3 days prior to the trial. The participants were placed in a bed in a supine position and two venous catheters were inserted in an antecubital vein in each arm and a background blood sample was taken. Hereafter, at − 270 min (see Fig. 1), a continuous infusion with L-[13C6] phenylalanine tracer (Cambridge Isotope Laboratories, Tewksbury, MA, USA) at an infusion rate of 6.0 µmol kg FFM−1 h−1 was started after injection of a priming dose 6.0 µmol kg FFM−1 over 2 min. The tracers were dissolved in sterile saline water and filtered through 0.20-µm-pore disposal filters (Minisart, Sartorius Stedium Biotech, Gottingen, Germany). The tracer infusion rate was set to obtain a venous tracer-to-tracee ratio (TTR) of ~ 10%. After reaching steady state at − 180 min, another blood sample and the first muscle biopsy were taken. The participants continued to rest in the supine position until another blood sample and biopsy were taken at 0 min. Immediately after, a drink containing 20 g of whey hydrolysate and 10 g of glucose was provided and finished immediately. Then blood samples were taken at 20 min, 40 min, 60 min, 90 min, 120 min and 240 min after the biopsy. At 240 min the last biopsy was taken, and the infusion stopped.

Fig. 1figure 1

Acute trial study protocol conducted at before and after the 12-month of intervention period

Blood samples

All blood samples were collected in 9 mL plasma Vacutainers containing EDTA, placed on ice for ≥ 10 min, and spun down at 3200 g for 10 min at 4 °C. Plasma was then transferred to Eppendorf tubes and stored at − 80 °C until further analysis.

Muscle biopsies

All three biopsies were obtained from the vastus lateralis with individual incisions with ~ 3 cm in between with a 4-mm biopsy needle (Bergström, Stockholm, Sweden) using manual suction. At the beginning of the trial, the skin was shaved, the thigh muscle was inspected and the incision sites for the three biopsies were marked. Before obtaining each biopsy, the area was disinfected and local anesthetic (1% lidocaine) was administered. A ~ 1 cm incision was made before inserting the needle and obtaining the biopsy. An elastic band with a compression pad was used to compress the incision site for 30 min to avoid intramuscular hematoma. Before compression, the incisions were strapped with SteaStrips and covered with waterproof plaster. The muscle specimens were quickly cleansed from any visible blood, fat and connective tissue under a microscope, and then frozen in liquid N2 and stored at − 80 °C until further analysis.

FSR

The myofibrillar protein fractional synthesis rate (FSR) was calculated for two periods (see Fig. 1) using the precursor–product model as illustrated:

$$\mathrm=\frac}_,\mathrm}}}_,\mathrm} \times t)} \times 100,$$

where \(}_,\mathrm}\) is the change in myofibrillar protein-bound phenylalanine enrichment between two consecutive biopsies with t hours in between; \(}_,\mathrm}\) is the plasma weighted mean phenylalanine enrichment between the two biopsies. The 3-h basal synthesis rate was calculated using the biopsies and blood samples at − 180 min and 0 min (abbreviated FSRbasal), and the 4-h synthesis rate in response to protein intake using the biopsies at 0 min and 240 min and a weighed mean of the plasma enrichment levels measured in the blood samples from 0, 20, 60, 90 and 240 min (abbreviated FSRresponse). A factor of 100 was used to express FSR in percent per hour (% h−1) [21]. The muscle specimens were prepared as follows: ~ 20 mg of the muscle sample was transferred to a 2-mL lysing tube containing 10 lysing beads and two silicon carbide crystals. One mL of 4 °C homogenizing buffer (Tris 0.02 M [pH 7.4], NaCl 0.15 M, ED(G)TA 2 mM, Triton X-100 0.5%, sucrose 0.25 M) was added and the sample was homogenized 4 ∙ 45 s at a speed of 5.5 m s−1 with a 2-min pause in between (FastPrep 120A-230; Thermo Savant, Holbrook, NY, USA). The samples were then rested for 3 h at 5 °C. They were then spun at 800 g for 20 min at 5 °C and the supernatant discarded. One mL of 4 °C homogenizing buffer were added to the pellet and the sample was once again homogenized for 1 ∙ 45 s at a speed of 5.5 m s−1, left for 30 min at 5 °C and then spun 800 g for 20 min at 5 °C. The supernatant was again discarded and 1.5 mL KCl buffer (KCl 0.7 M, pyrophosphate (Na4P2O7) 0.1 M) added and the samples were vortexed and left overnight at 5 °C. The sample was then vortexed and spun at 1600 g for 20 min at 5 °C and the supernatant (the myofibrillar protein fraction) was then transferred to a Scot glass and 2.3 mL ethanol 99% was added. The samples were then vortexed and left for 2 h at 5 °C. After a spin at 1600 g for 20 min at 5 °C, the supernatant was discarded and 1 mL 70% ethanol was added to the pellet containing the myofibrillar protein fraction. The samples were vortexed and then spun at 1600 g for 20 min at 5 °C and the supernatants were once again discarded. To hydrolyze the myofibrillar proteins, 1 mL of 6 M HCl was added and the sample left overnight at 110 °C. The constituent amino acids were then purified over Dowex resin (AG 50W-X8 resin; Bio-Rad Laboratories, Hercules, CA) columns using 2 M NH4OH for elution and put under N2 flow at 70 °C until dried. Hereafter, the amino acids were derivatized as the N-acetyl-propyl (NAP) derivative as described in detail previously [22]. After derivatization, the samples were analyzed using a gas chromatography combustion isotope ratio mass spectrometry (GC–C–IRMS) system (Hewlett Packard 5890-Finnigan GC combustion III-Finnigan Deltaplus; Finnigan MAT; Bremen; Germany). Briefly, 1 µL of sample was injected using a solvent split mode programmed-temperature vaporization (PVT) inlet. A detailed description of settings has been published previously [21]. The tracer enrichments in plasma were analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Plasma samples were prepared and analyzed as described by Bornø et al. 2014 [23].

Muscle metabolome

The muscle metabolome was measured using biopsies at time point 0 min and 240 min after the intake of cocktail, both at 0 month and after 12 months of intervention. Muscle samples were extracted using a similar method as described by Alves et al. 2015 [16], which is based on methanol/chloroform/water at Vol:Vol:Vol ratio of 1:1.2:1, respectively. The muscle specimens were prepared and analyzed as follows. ~ 25 mg of frozen muscle tissue was put into 2 mL lysing tubes containing 10 lysing beads (MP Blomedicals, Ohio, USA) and two silicon carbide crystals (Biospech products inc., Bartlesville, USA). Then 0.5 mL of ice-cooled 50% methanol, containing 20 ppm ribitol, was added. The biopsies were homogenized by stirring four times with an interval of 1 min at a speed of 5.5 m s−1 at 5 °C (FastPrep 120A-230, Thermo Savant, Holbrook, NY, USA) with a 2 min pause in between to avoid heating. Then, 300 µL of chloroform was added and the homogenized samples were vigorously vortexed for 10 min at room temperature. The samples were rested on ice for 20 min and centrifuged for 15 min at 5 °C at 16,000 g. Sixty µL of the upper part of the aliquot (methanol part) and 40 µL of the lower part of the aliquot (chloroform part) were put into 200 µL glass inserts. The glass inserts were then dried under reduced vacuum using a SpeedVac (Labogene, Lynge, Denmark) at 40 °C for 3 h. Samples were then derivatized in two steps, first by addition of 10 µL 20 mg mL−1 methoxamine hydrochloride in dry pyridine (90 min at 45 °C by agitating at 750 rpm) followed by trimethylsilylation (TMS) using trimethylsilyl cyanide (TMSCN), as described previously [24]. TMS derivatization was performed by addition 40 µL TMSCN and by agitating at 750 rpm for 40 min at 45 °C. A total of 226 samples were analyzed by GC–MS in a randomized order; 206 samples originate from this study design and 20 samples were controls consisting of pooled muscle samples run every 10th sample in the sequence.

Sample derivatization and injection of 1 µL derivatized aliquot were automated using a Dual-Rail MultiPurpose Sampler (MPS) (Gerstel, Mülheim an der Ruhr, Germany) as previously described [25]. The GC–MS consisted of an Agilent 7890B gas chromatograph (GC) (Agilent Technologies, California, USA) coupled with a HT Pegasus time-of-flight mass spectrometer (LECO Corporation, Saint Joseph, USA). A GC column used was Restek ZB 5% Phenyl 95% Dimethylpolysiloxane column (30 m length, 25 µm diameter and 0.25 µm of film thickness) with a 5 m inactive guard column (Phenomenex, Torrance, USA). A hydrogen generator, Precision Hydrogen Trace 500 (Peak Scientific Instruments Ltd, Inchinnan, UK) was used to supply a carrier gas at the constant column flow rate of 1.0 mL min−1. The initial temperature of the GC oven was set to 40 °C and held for 2 min, followed by heating at 12 °C min−1 to 320 °C and kept for an additional 8 min, making the total run time 33.3 min. A post-run time at 40 °C was set to 5 min. Mass spectra were recorded in the range of 45–600 m/z with a scanning frequency of 10 Hz, and the MS detector and ion source were switched off during the first 6.4 min of solvent delay time. The transfer line and ion source temperature were set to 280 °C and 250 °C, respectively. The mass spectrometer was tuned according to manufacturer’s recommendation using perfluorotributylamine (PFTBA). The MPS and GC–MS were controlled using vendor software Maestro (Gerstel, Mülheim an der Ruhr, Germany) and ChromaTOF (LECO Corporation, Saint Joseph, USA). The raw GC–TOF–MS data were processed using Statistical Compare toolbox of the ChromaTOF software (Version 4.50.8.0) with the following settings: the raw data were used without smoothing prior to peak deconvolution; the baseline offset was set to 0.8; the expected average peak width was set to 1.2 s; the signal-to-noise was set to ≥ 5; the peak areas were calculated using deconvoluted mass spectra; the common m/z ions of derivatization products were determined as 73, 75, and 147. Deconvoluted mass spectra were also used for peak identification using LECO-Fiehn and NIST11 libraries. The library search was set to return top 10 hits with EI-MS match of > 75% using normal-forward search and with a mass threshold of 20. Deconvoluted peaks were aligned across all samples using the following settings: retention time shift allowance of < 3 s, EI-MS match > 90%, mass threshold > 25 and present in > 90% of all pooled samples.

Statistical analysis

FSR were analyzed according to the published study protocol [19] with a one-way ANOVA on each intervention arm separately comparing the difference between delta fractional synthesis rates (the FSR in the resting 4-h postprandial period after ingestion of 20 g whey protein ‘response’ subtracted the resting overnight fasted FSR ‘basal’) at 0 month and 12 months (ΔΔ FSR = (12monthFSRresponse − 12monthFSRbasal) − (0monthFSRresponse—0monthFSRbasal)). Further, two-way mixed ANOVA test was performed on the 0-month data set on basal vs. response with sex as the between-group factor. All FSR statistical analyses were performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California, USA). FSR were analyzed as modified intention to treat (mITT) that completed the two trials irrespective of adherence and as per protocol (PP). To be included in the PP analysis, participants needed a supplement adherence > 75%, as well as > 65% and > 75% adherence to training for those assigned to HRTW and LITW, respectively. Further, participants who conducted the 12-month acute trial more than 14 days after their last training session were also excluded from the PP analysis. mITT analyses were conducted on participants who completed the study irrespective of adherence to the intervention. The muscle metabolome data were subjected to univariate and multivariate statistical analysis prior to investigating the possible effects according to the study design factors, including visit (0 m and 12 m), treatment (basal and response) and the intervention (CARB, COLL, WHEY, LITW and HRTW). Principal component analysis (PCA) [26] was performed prior to explore the muscle metabolome data and evaluate the overall systematic variation present in the data. ANOVA-simultaneous component analysis (ASCA) [27] with permutation test, as described previously [28], was used to study the significance of the study design factors and their explained variations. Single metabolite differences related to the design factors were analyzed using an ANOVA adjusted for multiple testing using false discovery rate (FDR) of 10%. Prior to PCA, ASCA and ANOVA, the muscle metabolome data were normalized to the internal standard (ribitol) peak area. The muscle metabolome data were mean centered (the mean of each peak was subtracted from the corresponding variable) and divided by its standard deviation, also called “auto scaling” prior to PCA and ASCA analysis. All data analyses were conducted using MATLAB ver. 2016b (The Mathworks, Inc. USA) and custom MATLAB scripts written by the authors.

Blinding

Randomization was done by an investigator not involved in interventions or not sensitive to blinding. Participants in the WHEY, COLL and CARB group were blinded to which supplement they received. Training was not blinded to the participants. Blinded researchers performed and analyzed the outcome measures.

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