Greater myofibrillar protein synthesis following weight-bearing activity in obese old compared with non-obese old and young individuals

The mechanisms through which obesity impacts age-related muscle mass regulation are unclear. In the present study, rates of integrated myofibrillar protein synthesis (iMyoPS) were measured over 48-h prior-to and following a 45-min treadmill walk in 10 older-obese (O-OB, body fat[%]: 33 ± 3%), 10 older-non-obese (O-NO, 20 ± 3%), and 15 younger-non-obese (Y-NO, 13 ± 5%) individuals. Surface electromyography was used to determine thigh muscle “activation”. Quadriceps cross-sectional area (CSA), volume, and intramuscular thigh fat fraction (ITFF) were measured by magnetic resonance imaging. Quadriceps maximal voluntary contraction (MVC) was measured by dynamometry. Quadriceps CSA and volume were greater (muscle volume, Y-NO: 1182 ± 232 cm3; O-NO: 869 ± 155 cm3; O-OB: 881 ± 212 cm3, P < 0.01) and ITFF significantly lower (m. vastus lateralis, Y-NO: 3.0 ± 1.0%; O-NO: 4.0 ± 0.9%; O-OB: 9.1 ± 2.6%, P ≤ 0.03) in Y-NO compared with O-NO and O-OB, with no difference between O-NO and O-OB in quadriceps CSA and volume. ITFF was significantly higher in O-OB compared with O-NO. Relative MVC was lower in O-OB compared with Y-NO and O-NO (Y-NO: 5.5 ± 1.6 n·m/kg−1; O-NO: 3.9 ± 1.0 n·m/kg−1; O-OB: 2.9 ± 1.1 n·m/kg−1, P < 0.0001). Thigh muscle “activation” during the treadmill walk was greater in O-OB compared with Y-NO and O-NO (Y-NO: 30.5 ± 13.5%; O-NO: 35.8 ± 19.7%; O-OB: 68.3 ± 32.3%, P < 0.01). Habitual iMyoPS did not differ between groups, whereas iMyoPS was significantly elevated over 48-h post-walk in O-OB (+ 38.6 ± 1.2%·day−1, P < 0.01) but not Y-NO or O-NO (+ 11.4 ± 1.1%·day−1 and + 17.1 ± 1.1%·day−1, respectively, both P > 0.271). Equivalent muscle mass in O-OB may be explained by the muscle anabolic response to weight-bearing activity, whereas the age-related decline in indices of muscle quality appears to be exacerbated in O-OB and warrants further exploration. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-023-00833-2.


Introduction
The precipitous age-related loss of skeletal muscle mass, quality, and strength ("sarcopenia") has welldocumented health consequences for older adults that are associated with a significant societal and economic cost [16].Alarmingly, the number of individuals presenting with this musculoskeletal disease is increasing exponentially with population ageing [16].Increasing adiposity also occurs with advancing age [31], resulting in an unfavourable ratio of fat to muscle that is associated with poor overall health, reduced functional capacity, and lower quality of life alongside heightened risk of metabolic syndrome, type II diabetes, and cancer [29].Importantly, the health complications arising from sarcopenia and obesity are thought to be multiplicative [2].Indeed, sarcopenia progression is accelerated in obese older individuals [12,33,48], while sarcopenic obesity (the confluence of sarcopenia and obesity) heightens the risk of many chronic diseases and all-cause mortality [37].
Sarcopenia is largely underscored by a blunted muscle protein synthesis (MPS) response to amino acid nutrition, termed "anabolic resistance" [34], which is exacerbated by the presence of obesity [5,50,61].Despite this, measures of absolute lean mass have been shown to be equivalent or greater in obese compared with non-obese older adults (e.g.[61].Although relative lean tissue mass may, in some cases, remain lower in obese individuals due to their larger body mass [52], these data suggest that obesity in older age may offer some partial protection for absolute levels of lean mass.However, the mechanisms that underpin this phenomenon remain unclear.MPS is highly responsive to contractile loading (e.g.resistance exercise) and exercise/physical activity is a potent stimulus for muscle remodelling and maintenance in older adults [1].In obese older adults, the requirement to move more inert mass during weight-bearing activities (e.g.walking as part of activities of daily living) could induce sufficient loading forces to stimulate MPS and offset potential age-related muscle mass loss caused by anabolic resistance to amino acids [48,55], which may counteract amino acid-induced anabolic resistance to explain equivalent or greater absolute muscle mass in obese vs. non-obese older individuals.
While the loss of muscle mass is significantly related to loss of strength and function [73], the agerelated decline in muscle strength and, therefore, quality (strength/function relative to muscle mass) may occur more rapidly than the loss of muscle mass [24,42,44].The deterioration in muscle quality may be more apparent in obese older individuals, potentially through lipid-induced alterations to muscle contractile properties [63] and a requirement to move greater body mass [48,68], although not all studies support this notion [33].Importantly, muscle quality is strongly associated with numerous health outcomes [36] and strength/function has been consistently associated with lower all-cause mortality risk in later life (e.g.[58].While the presence of obesity in older age may be associated with equivalent or greater absolute muscle mass compared with non-obese older individuals, muscle quality may be worsened.To date, the "trade-off" between muscle mass and indices of muscle quality in obese older individuals has not been fully explored and requires clarification.Studies to date have also largely been limited to dual-energy x-ray absorptiometry measures lean tissue mass rather than muscle mass, per se (e.g.[61], Tomlinson, Erskine, Winwood, et al. 2014a).
The primary purpose of this study was to investigate rates of integrated myofibrillar protein synthesis (iMyoPS) prior-to and following a weight-bearing walking task in older obese (O-OB) compared with older non-obese (O-NO) and younger non-obese (Y-NO) individuals.Quadriceps muscle mass, intramuscular fat, maximal strength, and voluntary activation were measured as indices of muscle quality in O-OB, Y-NO, and O-NO.Our primary hypothesis was that the iMyoPS response to weight-bearing activity would be greater in O-OB compared with O-NO and Y-NO, with no differences between O-NO and Y-NO.In addition, we hypothesised that the iMy-oPS response to weight-bearing activity would be significantly greater compared with habitual free-living iMyoPS in O-OB but not O-NO and Y-NO.Lastly, we posited that quadriceps muscle mass would be equivalent or greater, intramuscular fat greater, and indices of muscle quality poorer in O-NO compared with Y-NO and exacerbated in O-OB.occasions over a ~ 3-week period (Fig. 1).For visits 3-7, described below, participants reported to the lab in an overnight fasted state at ~ 08:00 h (± 2 h).In addition, participants were asked to refrain from any strenuous physical activity and alcohol consumption throughout the iMyoPS assessment period (Fig. 1).

Visit 1: enrolment and eligibility
Following a telephone screening, participants reported to the CRF.On arrival, participants completed a general health questionnaire verified by a medical professional.
Anthropometrics and compartmental body composition (including whole-body skeletal muscle mass) were assessed, the latter via bioelectrical impedance analysis using a segmental body composition analyser (BC-418, Tanita Europe BV, Amsterdam, The Netherlands) incorporating assessments of segmental fat mass, body fat percentage, fat-free mass, and predicted skeletal muscle mass to verify eligibility and to stratify participants into respective groups.Finally, an SPPB was conducted to assess participants' function.The SPPB is a series of measures that combines the results of gait speed, chair stand, and balance tests, and typically used as a predictive tool for possible disability in older people.) from the right leg tibial tubercle through to the anterior-inferior iliac spine, with participants in a supine position on the scanner bed for assessment of thigh CSA, thigh muscle volume, and ITFF.Participants entered the magnet feet first, with multiple 16-channel body coils positioned over their thighs and patella.A 3-plane localiser scan was used to prepare the fast-spin-echo scans using the DIXON sequence.The total number of 3.00 mm images acquired was ~ 192 but varied depending on the length of the participants' thighs.Immediately following transverse plane imaging of the thigh, while lying in the same position, sagittal plane sections of the right knee were collected (slice thickness: 3.00 mm, TR: 500 ms, TE: 10 ms, flip angle: 124°) to measure patella tendon moment arm length for the assessment of in-situ muscle-specific force.Finally, participants returned to the laboratory for an assessment of isometric leg strength and voluntary activation (VA) of the right quadriceps, determined via isokinetic dynamometry (Biodex System 3, Shirley, NY, USA), as previously described [49].Briefly, participants completed 3 × 3 s MVC of the knee extensors with each contraction separated by a 60 s passive recovery period.
Vol:. ( 1234567890) During MVCs, single peripheral nerve stimulation pulses (100 µs pulses at 400 V) were triggered to occur as soon as a peak torque was achieved to provide a "superimposed twitch" (sTw).The stimuli were also delivered 2 s after the cessation of the MVC to provide a potentiated "resting twitch" (pTw).Electrical stimulation was applied by a constant current stimulator (Digitimer Stimulator DS7, Digitimer, UK).Prior to discharge, participants were provided with daily D 2 O "top-ups" (0.3 mL•kg of lean body mass −1 ) for the duration of the study to consume on waking for the next 7 consecutive days to ensure maintenance of a "steady state" of ∼ 0.2% APE deuterium enrichment of the body water pool throughout study involvement.
Participants were also supplied with sterile containers to provide daily sample prior to consumption of their daily dose of D 2 O for later analysis of 2 H body water enrichment.

Visits 4, 6, and 7: percutaneous skeletal muscle biopsy
Three days after visit 3, participants returned to the laboratory having provided a saliva sample and consumed the daily dose of D 2 O. Saliva samples were temporarily stored in the participant home fridge (3-5 °C) and returned to the investigator at each experimental visit, where they were subsequently centrifuged (11,000 g for 10 min at 4 °C) to separate salivary debris before storage at -80 °C until later analysis.Thereafter, an initial skeletal muscle biopsy from the vastus lateralis and venous blood samples were obtained, as previously described [61].Prior to departure, participants were provided with a "weight maintenance" food parcel (∼ 50% carbohydrate, ∼ 30% fat, and ∼ 20% protein) for the subsequent 4 days to standardise dietary conditions between groups during the period of iMyoPS assessment (Fig. 1, Supplemental Table 2).Energy requirements were calculated using the Harris-Benedict equation [27] multiplied by an activity factor which best reflected individual habitual activity patterns (range 1.2-1.9,but generally 1.35 for all groups).Participants could choose from a selection of food options and consisted of breakfast, lunch, dinner, and snacks to ensure regular intakes of protein throughout the day.Fruit juices were also provided and water permitted ad libitum.Participants then reported to the CRF 48 h later to complete a 45-min treadmill walk (described below).
Visits 6 and 7 were completed 24 h and 48 h after visit 5, respectively, and replicated the experimental procedures detailed in visit 4.

Visit 5: 45-min treadmill walk
To determine the impacts of a weight-bearing task on skeletal muscle properties, participants completed a 45-min treadmill walk at ~ 55% of VȮ 2peak .This intervention was selected to mimic the demands of a brisk walking activity completed as part of typical activities of daily living.A similar intensity-duration treadmill walk task was previously shown to modestly increase MPS above basal rates in non-obese older adults [65], which we expected might increase more robustly when performed by O-OB.Briefly, the treadmill speed and gradient were adjusted to ensure a constant intensity throughout the 45 min, with participants wearing the mouthpiece assembly, as described in visit 2, at intermittent periods to monitor oxygen consumption.During treadmill walking, participants wore shorts fitted with textile EMG electrodes to quantify quadriceps muscle and wholethigh muscle activation patterns (MShorts-AllSport, MyonTec Ltd, Finland).The EMG shorts were calibrated against maximal EMG achieved during visit 2.
Prior to and immediately post treadmill walk, skeletal muscle biopsies from the vastus lateralis and venous blood samples were obtained.Prior to discharge, participants consumed a post-exercise beverage containing ~ 23 g of whey isolate protein (Myprotein, Northwich, UK) to augment exercise-induced MPS similarly in the initial hours of recovery.

Integrated myofibrillar fractional synthetic rate and intramuscular signalling
Habitual (visits 4 and 5) and post-walk (visits 5-7) fractional protein synthetic rates (FSR) were determined by measurement of deuterium enrichment in the myofibrillar protein pool via gas chromatographypyrolysis-isotope ratio mass spectroscopy.On extraction of all muscle tissue, biopsy samples were quickly rinsed in ice-cold saline and blotted to remove any visible blood, fat, and connective tissue before being immediately snap frozen in liquid nitrogen and stored at -80 °C until analysis.The reader is directed to our Vol.: (0123456789) previous work which provides more detailed description of our isotope tracer protocol and experimental approaches to assess body water enrichment and myofibrillar protein enrichment (i.e.assessment of myofibrillar bound 2 H-alanine) [43].Alanine was used as the amino acid for analysis due to its abundance, multiple sites of incorporation (e.g.muscle, plasma, saliva), and close representation of the total amino acid pool.Therefore, FSR was calculated from the enrichment of alanine, as calculated from body water enrichment, and the enrichment of alanine in the myofibrillar proteins and extrapolated to the entire amino acid pool.Briefly, ~ 30 mg of tissue was rapidly homogenised prior to separation of the myofibrillar-enriched portion from mitochondrial, collagen, and sarcoplasmic muscle fractions.Once the myofibrillar-enriched fraction had been washed and purified, proteinbound amino acids were released using acid hydrolysis and left to hydrolyse at 110 °C overnight, before being eluted from resin with ammonium hydroxide (NH 4 OH) on cation-exchange columns.Thereafter, samples were dried and reconstituted in hydrochloric acid (HCl) before analysis by gas chromatographypyrolysis-isotope ratio mass spectroscopy (Metabolic Solutions, Nashua, NH, USA).Anabolic (i.e.mTORmediated signalling) intramuscular signalling markers were determined for total (4E-BP1, AMPK, Akt, rpS6, IRS-1, TSC2) and phosphorylation (4E-BP1 Thr37/46 , AMPK Thr72 , Akt Serine 473 , rpS6 Ser235/236 , IRS-1 Ser636/639 , TSC2 Thr1462 ) protein content by western blot analysis on the sarcoplasmic protein fraction obtained during myofibrillar protein isolation, as previously described (e.g.[43,61] (see supplementary methods for primary antibodies for immunoblotting).
Calculation of fractional synthetic rate iMyoPS was calculated using the standard precursorproduct method, from the incorporation of deuterium-labelled alanine into protein, using the enrichment of body water (corrected for the mean number of deuterium moieties incorporated per alanine and the dilution from the total number of hydrogens in the derivative) as the surrogate precursor labelling between subsequent biopsies.FSR was calculated as follows: where APE Ala is deuterium enrichment of proteinbound alanine, APE P is mean precursor enrichment over time, and t is the time between biopsies.Briefly, ROIs were manually drawn around each respective muscle prior to analysis of signal intensity at the same location for water-only and fat-only captured images.ROIs were drawn in three consecutive slices at 50% limb length in the same manner, with the three values averaged for each participant.Due to issues pertaining to contraindications associated with MRI scans and the impact of COVID on in-person research activity, MRI outputs consisted of 29 datasets distributed within the groups as follows: 15/15 (Y-NO), 7/10 (O-NO), and 7/10 (O-OB).For assessment of quadriceps muscle force, for each 3 s contraction, maximal voluntary force was determined as the mean value over a 1 s period, which approximated the plateau level of the highest torque.The resting twitch torque was calculated as the peak torque achieved following the single pulse delivered 1 s post-MVC.The superimposed twitch torque was calculated as the increment in torque immediately following the pulse during MVCs.Voluntary activation (VA) was calculated using the interpolated twitch method.Specifically, the increment in torque (i.e.superimposed twitch) evoked during the MVCs was expressed as a fraction of the amplitude of the resting potentiated twitch produced with the same (1) Fat fraction(%) = 100 × Fatmeanintensity Water mean intensity + Fat mean intensity stimuli in the relaxed muscle post-MVC.The level of voluntary drive was then quantified as a percentage as previously described [47].Finally, dietary intake was analysed via dietary records using DietPlan 7 software (Forestfield Ltd, West Sussex, UK).

Statistical analysis
Differences in baseline group characteristics and quadriceps muscle volume characteristics were assessed via one-way analysis of variance (ANOVA).Differences in integrated myofibrillar protein synthesis (iMyoPS) and intramuscular signalling were assessed using a mixed model ANOVA with one within (two levels-48-h habitual and 48-h exercise induced) and one between factor (three levels; group [Y-NO, O-NO, O-OB]).
Where the ANOVA revealed a significant effect, posthoc analysis was conducted using a Holm-Bonferroni correction to isolate specific differences.For calculation of effect size, partial eta squared (η 2 ) was used for omnibus tests, where η 2 = 0.01, 0.06, and 0.14 indicate small, medium, and large effects, respectively.Cohen's d was used to calculate the effect size for post-hoc comparisons, where d = 0.2, 0.5, and 0.8 indicate small, medium, and large effects, respectively.Where sphericity was violated, a Greenhouse-Geisser correction factor was used.Normal distribution was assessed using the Shapiro-Wilk test.Where appropriate, non-normally distributed variables were logarithmically transformed.All other data were analysed using a one-way ANOVA.For all the tests, results were considered statistically significant when P < 0.05.Data are presented as mean ± standard deviation or standard error of the mean, unless otherwise indicated.All statistical analyses were conducted using IBM SPSS Statistics version 28.

Physical activity, diet, and blood characteristics
Anthropometrics, SPPB score, body composition, and metabolic health data for Y-NO, O-NO, and O-OB can be seen in Table 1.Body mass, BMI, whole-body fat mass, and leg fat mass were significantly higher in O-OB compared with O-NO and Y-NO (all P < 0.001), with no differences in body mass (P = 0.346, d = 0.41) and BMI (P = 0.136, d = 0.65) between Y-NO and O-NO (Table 1).Whole-body fat free mass and skeletal muscle mass were lower in O-OB and O-NO compared with Y-NO (all P < 0.001, d > 0.98) and were lower in O-OB compared with O-NO (P < 0.01; d > 1.52, Table 1).Plasma IL-6 and CRP were significantly elevated in O-NO (both P < 0.001, d > 1.40) and O-OB (both P < 0.001, d > 1.93) compared with Y-NO and greater in O-OB compared with O-NO (both P < 0.01, d = 0.81).HOMA-IR, fasting plasma glucose, serum insulin, and plasma HbA1C were significantly elevated in O-OB compared with Y-NO (Table 1, all P < 0.028, η 2 > 0.26), but did not differ between O-NO and Y-NO.All participants achieved a score of ≥ 9 in the SPPB (Table 1), demonstrating a good level of physical function.Habitual physical activity characteristics and self-reported dietary intake are presented in Table 2. Average daily step count was significantly higher in Y-NO (P < 0.01, η 2 = 0.33) compared with O-NO (P = 0.035, d = 0.92) and O-OB (P < 0.001, d = 1.70).
Step count on the day of the treadmill walk (visit 5) was significantly elevated above all other days in O-NO and O-OB (P < 0.001) but not Y-NO (P = 0.986, Fig. 2).3).Representative cross-sectional images at 50% thigh length for each group are presented in Fig. 3D.

Discussion
Age-related muscle loss is typically accompanied by a concomitant increase in whole-body adiposity and ectopic fat deposition within skeletal muscle [3,23,25].Skeletal muscle anabolic resistance to amino acids, which contributes to age-related muscle loss, is exacerbated by obesity through an impaired metabolic phenotype, accumulation of intramyocellular lipids (myosteatosis), dyslipidaemia, and adipose tissue-derived inflammation [5,13,23,50,56,61,63].In addition, obesity is also typically associated with sedentarism, reduced physical activity, and poor diet quality [68], factors which accelerate age-related muscle deterioration.Despite this, absolute levels of lean mass have been shown to be equivalent or greater in older obese compared with older non-obese individuals [50,61].Conversely, the age-related decline in muscle quality appears to be aggravated by the presence of obesity [22,32,35,52,59].We hypothesised that equivalent or greater absolute muscle mass in older obese (i.e.O-OB) compared with non-obese older (i.e.O-NO) and younger (i.e.Y-NO) The present data demonstrate that rates of integrated myofibrillar protein synthesis (iMyoPS) were increased above habitual values over 48-h after a 45-min weightbearing walking task in O-OB only (39%), but not Y-NO and O-NO (11% and 12%, respectively).Although we may have been underpowered to detect a significant interaction effect in the iMyoPS response to the walking task, the large effect size for both absolute and delta change in iMyoPS for O-OB only indicates significantly elevated rates in this cohort.The iMyoPS response to weight-bearing activity in O-OB was likely due to the mechanical stimulus of a greater loading force (i.e.moving more inert mass and a greater absolute body mass) as previously postulated [28,48].Indeed, quadriceps muscle "activity', assessed using average surface EMG amplitude across the 45-min task, was significantly greater in O-OB compared with Y-NO and O-NO (68% of maximal EMG amplitude compared with 31% and 36% in Y-NO and O-NO, respectively).These data are consistent with previous observations suggesting an increase in muscle contractile work for obese individuals when walking and greater load carriage of weight-bearing muscles (e.g.[54].That diet and physical activity were kept constant during iMyoPS assessment also suggests that the increase in iMyoPS in O-OB was a direct response to the weight-bearing walking task.It is also worth noting that, although daily step count was greater than habitual values on the day of the 45-min walk in O-OB and O-NO, iMyoPS increased above habitual rates in O-OB only.The discrepancy between the current iMyoPS data and previous findings of a similar or impaired muscle anabolic response to muscle mechanical contraction in young and middle-aged obese individuals [4,26,30] is likely due to differences in muscle contractile loading between seated resistance or aerobic exercise and weight-bearing activity.Nonetheless, it appears that obesity impairs muscle adaptive remodelling to traditional exercise training modalities, independently of ageing.Given that O-OB in this study were relatively active (~ 8500 daily steps on average) and lower-risk obese (~ 31 kg•m −2 , 33% body fat), we cannot rule out that the iMyoPS response to weight-bearing activity is diminished in more sedentary and/or severely O-OB individuals.The physical activity status of O-OB also supports the notion that the increase in iMyoPS following weight-bearing activity is not a response to damage, degradation, and/or synthesis imbalances through exposure to an unaccustomed task (K.E. [7,18,57].
Previously, we have demonstrated that the acute myofibrillar MPS response to moderate-dose protein ingestion at rest was diminished in O-OB compared with Y-NO and O-NO, and was associated with leg fat mass and physical activity levels [61].Resistance exercise contraction (e.g.[20] and moderate-intensity treadmill walking (e.g.[65] have been shown to augment postprandial muscle anabolism in young and older adults (i.e.alleviate anabolic resistance) by increasing the use of dietary-derived amino acids for MPS (e.g.[55].Furthermore, resistance exercise and load-carriage can stimulate an increase in postabsorptive MPS (e.g.[54].Hence, it is possible that the observed increase in iMyoPS rates in O-OB over 48 h after weightbearing physical activity may be underscored by augmented postabsorptive and/or postprandial MPS, although our study design did not enable us to determine this.Notwithstanding, the measure of iMyoPS using D 2 O ingestion is a less invasive alternative to acutely measured MPS protocols, which determines muscle anabolism under "free-living" conditions, incorporating all feeding and physical activity events, and is associated with longer-term muscle mass change in older adults [10,11].
Interestingly, habitual iMyoPS rates over 48 h prior to the walking task did not differ between groups.Given earlier evidence of impaired postprandial MPS in O-NO and O-OB compared with Y-NO [5,50,61], and the notion that cumulative deficits in MPS drive the progression of sarcopenia [10,11], it would be reasonable to expect habitual iMyoPS to be lower in O-OB and O-NO compared with Y-NO.It is possible that the diet and activity control measures implemented during the assessment of iMyoPS and/or the relatively short 48-h timeframe of assessment could have impacted our ability to detect subtle between-group differences in iMyoPS.We also speculate that the degree of weightbearing activity in O-OB over the 48-h habitual phase may have provided sufficient stimulus to counteract any postprandial muscle anabolic resistance, thereby eliciting habitual iMyoPS rates that were indistinguishable from O-NO.Nonetheless, acute postabsorptive and postprandial myofibrillar fractional synthesis rates on the order of 0.02-0.06%•h−1 have been widely reported in young and older individuals (e.g.[46,61], suggesting the present habitual "free-living" iMyoPS rates of 0.6-1.4%•day−1 are within the expected physiological range.Given the potential importance of altered muscle protein breakdown with ageing and obesity [17,50,66], this locus of muscle mass regulation warrants further investigation.Similarly, our analysis of rested and immediate post-walk intramuscular signalling regulators of MPS revealed no differences within or between groups, suggesting that more detailed temporal signalling work may be required to understand the molecular regulators of muscle mass in O-OB. Vol.: (0123456789) An increased iMyoPS response to weight-bearing activity in O-OB, in the face of any postprandial muscle anabolic resistance (i.e. in response to amino acid nutrition), may provide the mechanistic basis for the equivalent absolute quadriceps muscle volume/CSA between O-OB and O-NO.That absolute muscle volume and CSA was not greater in O-OB compared with O-NO may be due to the low-risk obesity status in this cohort or variability in our relatively small sample.Indeed, others have shown greater muscle mass (and markers of) in more severely obese older adults (e.g.[70][71][72].Although not determined herein, it is likely that muscle fibre CSA may have been greater in O-OB compared with O-NO, as we have previously demonstrated [61].Importantly, the brisk walk task in the current study increased step-count well above habitual levels in O-OB.Therefore, it is unlikely that O-OB would regularly stimulate iMy-oPS rates to the same robust levels observed over 48-h post-walk task that would theoretically promote greater muscle mass in O-OB over O-NO.Furthermore, relative daily protein intake for O-OB (0.94 g⋅ kg −1 ) was also below recommendations for longerterm muscle retention in older adults [51], which would likely be deleterious to muscle mass accretion, particularly in the face of postprandial muscle anabolic resistance in O-OB.Knowledge of whether muscle mass in non-load bearing regions is equivalent or greater in O-OB over O-NO would provide clarity to our hypothesis that weight-bearing activity induces muscle anabolism and supports absolute muscle mass in O-OB.Any such future work would also have important implications for other basic physiological functions (e.g.breathing, lifting, mobilising, eating) that may decline rapidly in the presence of the obese metabolic phenotype [14,60].While our data provide a valuable contribution to our understanding of the relationship between obesity, ageing, and muscle regulation, these data should be interpreted cautiously as it is not known how persistent this effect may be in older obese men and/or if they are present in women.Indeed, the effects of obesity on muscle protein turnover in pre-sarcopenic middle-aged, severely obese as well as frail (i.e.exhibiting very poor function), and oldest-old males and females need to be comprehensively assessed.
Muscle quality, defined as strength and/or power per unit of muscle mass, is lost more rapidly than absolute muscle mass with advancing age [24,42] and represents a significant contributor to the impairment in physical function [42,64], particularly in obese individuals with a larger body mass to carry.Indeed, it is generally accepted that obese individuals, independent of age, have lower maximal strength when expressed relative to body mass (e.g.[39,50].In the present study, no differences in absolute MVC were observed between O-NO and O-OB, though age-related impairments were found.However, MVC relative to total body mass was lower in O-NO compared with Y-NO and was exacerbated further in O-OB, consistent with previous findings (e.g.[50,62,[69][70][71], Tomlinson, Erskine, Winwood, et al. 2014a).The difference between O-NO and O-OB in MVC relative to peak quadriceps volume/CSA was not statistically significant, which may be due to the relatively low sample size and large variability in relative MVC measures.Nonetheless, a large effect size (d = 0.86) in MVC/peak CSA was apparent between O-OB and O-NO.Indeed, previous studies demonstrate that the age-related decline in muscle quality, assessed via markers of in-situ muscle-specific force, is exacerbated by obesity, even in the presence of increased strength and muscle volume [67], Tomlinson, Erskine, Winwood, et al. 2014a, 2014b;[72].This is important as reduced relative lower-limb strength can manifest with impaired functional capacity (e.g.difficulty walking, climbing stairs, rising from a chair/bed) [38], and increased risk of joint pathologies [15], that would have a profound impact on overall quality of life in O-OB.Interestingly, quadriceps voluntary activation did not differ between groups, which contrasts with previous studies suggesting that obesity is associated with impaired voluntary recruitment of muscle (e.g.[8,9,40,53].The relatively low-risk obese and high functional status of O-OB in the present study may have impacted our ability to detect a clear deficit in voluntary activation.Indeed, in more advanced metabolic disease and severe O-OB individuals, declines in muscle functional quality and voluntary activation appear to be more pronounced (J. A. [6,19,68],Tomlinson, Erskine, Winwood, et al. 2014a, 2014b).Collectively, our findings provide some evidence of lower muscle functional quality with ageing that may be exacerbated by the presence of obesity.The driving mechanisms of lowered muscle quality in O-OB may be partly modulated by lipid-induced alterations, including systemic inflammation, intracellular lipotoxicity, impaired muscle structure/contractility, or impaired skeletal muscle regenerative capacity (for review [48], which warrants further exploration.
Infiltration of fat within skeletal muscle is apparent with ageing and exacerbated by the presence of obesity via lipid overspill, with implications for metabolic health and muscle structure/contractility [3,22,23,25,62,63].In the present study, quadriceps ITFF, a marker of fat infiltration within the muscle, was significantly elevated in O-NO compared with Y-NO and even greater in O-OB, which is consistent with previous findings [21,41,45].Although we did not directly measure intramyocellular lipid content at a microscopic level, we previously reported a robust two-fold greater type II fibre lipid infiltration O-OB compared with O-NO that coincided with greater postprandial muscle anabolic resistance [61].Intuitively, the greater ITFF in O-OB compared with O-NO would partly explain their worsened metabolic phenotype (markers of insulin resistance and systemic inflammation) and signs of poorer muscle functional quality.Considering the metabolic and functional health status of our O-OB cohort relative to more severe O-OB cohorts included in previous studies (e.g.[70][71][72], it is possible that differences in muscle functional quality would diverge further from O-NO as metabolic disease progresses to an advanced stage [2,[68][69][70][71].Whether the abundance, species, or sub-cellular location of intramuscular lipids influences indices of muscle quality in O-OB has yet to be explored.Collectively, the equivalent or greater absolute muscle mass in O-OB over O-NO is concurrent with greater intramuscular fat, which has dire consequences for muscle function, metabolic health, and overall quality of life.
In conclusion, we demonstrate here that weightbearing activity stimulated an increase in 48-h iMy-oPS rates above habitual values in O-OB, but not O-NO and Y-NO.These data may explain observations of equivalent or greater muscle mass in O-OB compared with O-NO and Y-NO, with weight-bearing activity potentially counteracting acute postprandial muscle anabolic resistance previously shown in O-OB.In line with the worsened metabolic phenotype and greater ITFF of O-OB, the present data suggests that the age-related decline in indices of muscle quality may be aggravated by the presence of obesity, the mechanistic basis of which requires further attention.Finally, the effects of obesity on muscle protein turnover in pre-sarcopenic middle-aged, severely obese, as well as frail and oldest-old males and females need to be comprehensively assessed to develop feasible and effective interventions for fat mass reduction and muscle mass maintenance/growth that will combat rising sarcopenic-obesity prevalence.

Fig. 2
Fig. 2 Step count during the 7-day MPS assessment period for young non-obese (Y-NO; dark grey circles), old non-obese (O-NO; light grey circles), and old obese (O-OB; clear circles) adults.The 48-h habitual (i.e., pre-walk) and 48-h exercise (i.e.post-walk) phases are shaded in grey.Average daily step count was significantly higher in Y-NO compared with O-NO and O-OB.Step count on day 5 was significantly elevated, above habitual levels, in O-NO and O-OB but not Y-NO.Significance was set at P < 0.05.* Significantly different from O-NO; # significantly different from O-OB.Values are presented as means ± SD

Fig. 3
Fig. 3 Quadriceps muscle volume (A) and peak quadriceps muscle cross sectional area (CSA, B) for young non-obese (Y-NO, dark grey bars), old non-obese (O-NO, light grey bars), and old obese (O-OB, white bars), respectively.Quadriceps muscle intramuscular thigh fat fraction (ITFF) for Y-NO, O-NO, and O-OB can be seen in C, respectively, for vastus lateralis (VL, dark grey boxes), vastus medialis (light grey boxes), and rectus femoris (white boxes).Values are presented as the median (central horizontal line), 25th and 75th percentiles (box), minimum and maximum values (vertical lines), and mean (cross).Representative CSA images at 50% of thigh length can be seen in D for Y-NO, O-NO, and O-OB, respectively.The young non-obese individual exhibits a larger quantity of skeletal muscle mass, whereas high infiltration by adipose tissue is observed in the older obese individual.Significance was set at P < 0.05.For A and B, * Significantly different from O-NO, # significantly different from O-OB.For C, * VL significantly different from

Fig. 4
Fig. 4 Normalised quadriceps maximal voluntary contraction force (MVC, A), quadriceps MVC relative to body mass (BW, B), quadriceps MVC relative to quadriceps peak cross-sectional area (CSA, C), and quadriceps MVC relative to quadriceps muscle mass (D) for young non-obese (Y-NO, dark grey boxes), old non-obese (O-NO, light grey boxes), and old obese

Fig. 5
Fig. 5 Integrated myofibrillar protein synthesis (iMyoPS) fractional synthetic rates (FSR) over 48 h in the habitual state (black bars) and following a 45-min treadmill walk at ~ 55% VȮ 2peak (white bars) in young non-obese (Y-NO), old nonobese (O-ON), and old obese (O-OB), respectively (A).Values are presented as means ± SEM.Individual responses are shown by grey circles and grey squares for pre-and post-walk values, respectively.Delta change in integrated myofibrillar protein fractional synthesis rates (FSR) from habitual to 48 h-post-exercise is presented in B. Average electromyography (EMG) amplitude, a marker of thigh muscle activity, during the 45-min treadmill walk is presented in C and expressed

Table 1
Anthropometric, SPPB, body composition, and metabolic health dataData presented as mean ± standard deviation.SMM (%) presented relative to total body weight.
* Significantly different from Y-NO, # significantly different from O-NO Abbreviations: Y-NO, young non-obese; O-NO, older-non-obese; O-OB, older obese; HOMA-IR, homeostasis model assessment of insulin resistance; FFM, fat free mass; SMM, skeletal muscle mass; SPPB, short physical performance battery; AU, arbitrary units; RMR, resting metabolic rate Y-NO, O-NO, and O-OB were recruited to investigate the role of obesity on rates of integrated myofibrillar protein synthesis (iMyoPS) and indices of muscle mass, quality, function, and morphology.Participants reported to the National Institute for Health Research/Wellcome Trust Clinical Research Facility (CRF) of the Queen Elizabeth Hospital (Birmingham, UK), the Centre for Human Brain Health ([CHBH] University of Birmingham, UK), and/ or the Sport, Exercise and Rehabilitation Sciences Laboratory (University of Birmingham, UK) on 7 from the distal end of the femur working proximally) were excluded so that only the middle 50% region of the assessed area was used for manual segmentation.These CSA were summed and multiplied by the distance between slices to estimate quadriceps-specific and total thigh muscle volume.In addition, peak CSA and CSA at 50% (of femur length) were calculated.All analyses were conducted by the same investigator (PM), and every 10 slices were blind checked for quality control (JQ), with an interrater agreement of 0.97.For quadriceps-specific and total thigh intramuscular lipid quantification (i.e.ITFF), images acquired with a 2-point Dixon MRI sequence were analysed using Medical Image Processing, Analysis and Visualization ([MIPAV] National Institutes of Health, Bethesda, MD, USA) software to isolate regions of interest (ROI).The Dixon method is an MRI sequence based on chemical shift and designed to achieve uniform fat suppression.The Dixon technique exploits the fact that water and fat molecules were process at different rates(Dixon et al. 1984).A 2-point Dixon acquisition exploits the difference between resonance frequencies of hydrogen nuclei in water and fat molecules to produce four sets of images: water only, fat only, in phase, and out of phase.Calculation of fat fraction (%) was carried out blinded using the following formula: via an A/D converter.The 1000 Hz signal was first rectified and then averaged over non-overlapping 100 ms intervals, similar to the root mean square method.The averaged data was stored in the memory of the MCell at 25 Hz, from which the data was extracted and analysed using customised software (Muscle Monitor, Myon-Tec Ltd, Kuopio, Finland).All EMG data was visually assessed and corrected for artefacts.The maximal EMG signal for visit 2 was determined as the highest 30 s mean value.For visit 5, to obtain an average rectified value of EMG, the average EMG signal was taken from the