The gradual deterioration of physiological systems with ageing makes it difficult to maintain skeletal muscle mass (sarcopenia), at least partly due to the presence of 'anabolic resistance', resulting in muscle loss. Sarcopenia can be transiently but markedly accelerated through periods of muscle disuse-induced (i.e., unloading) atrophy due to reduced physical activity, sickness, immobilisation or hospitalisation. Periods of disuse are detrimental to older adults' overall quality of life and substantially increase their risk of falls, physical and social dependence, and early mortality. Disuse events induce skeletal muscle atrophy through various mechanisms, including anabolic resistance, inflammation, disturbed proteostasis and mitochondrial dysfunction, all of which tip the scales in favour of a negative net protein balance and subsequent muscle loss. Concerningly, recovery from disuse atrophy is more difficult for older adults than their younger counterparts. Resistance training (RT) is a potent anabolic stimulus that can robustly stimulate muscle protein synthesis and mitigate muscle losses in older adults when implemented before, during and following unloading. RT may take the form of traditional weightlifting-focused RT, bodyweight training and lower- and higher-load RT. When combined with sufficient dietary protein, RT can accelerate older adults' recovery from a disuse event, mitigate frailty and improve mobility; however, few older adults regularly participate in RT. A feasible and practical approach to improving the accessibility and acceptability of RT is through the use of resistance bands. Moving forward, RT must be prescribed to older adults to mitigate the negative consequences of disuse atrophy.

Objective Nutritional intervention studies have indicated that whey protein- and leucine-enriched multi-nutrient formulas high in vitamin D3 are optimal medical nutrition recipes for treating sarcopenia. The present study undertakes a scoping review of research on whey protein- and leucine-enriched multi-nutrient formulas high in vitamin D3 in older adults with sarcopenia to highlight the impact of feeding and feeding with exercise on body composition, strength, and physical function. Design Online databases identified studies published from 2005-2023, for which we selected 11 English-language studies that experimented with a whey protein—and leucine-enriched multi-nutrient formula(e) high in vitamin D3 on measures of sarcopenia as the primary outcome variables in older adults diagnosed as sarcopenic. Results Nine registered human clinical trials and two unregistered human clinical trials were included in the analysis. All studies included sarcopenic older adults ages 66.5 y to 86.5 y. Two intervention types were identified: nutrition only or nutrition + exercise rehabilitation. The nutritional interventions improved lean mass, strength, and physical function. Conclusion There is evidence for the effectiveness of several types of whey protein- and leucine-enriched multi-nutrient formulas high in vitamin D3 in improving lean mass and physical performance in older adults recovering from sarcopenia. This is the first scoping review to show that specific formula(e) used with rehabilitation programs can alleviate sarcopenic obesity, support skeletal bone, and improve body composition, lean mass, and physical function with or without exercise (CRD42022342953).

The effects of supplementation with whey protein alone or with vitamin D on sarcopenia-related outcomes in older adults are unclear. We aimed to assess the effect of whey protein supplementation alone or with vitamin D on lean mass (LM), strength, and function in older adults with or without sarcopenia or frailty. We searched PubMed, Web of Science, and SCOPUS databases. Randomized controlled trials (RCT) that investigated the effect of whey protein supplementation with or without vitamin D on sarcopenia outcomes in healthy and sarcopenic or frail older adults were included. Standardized mean differences (SMDs) were calculated for LM, muscle strength, and physical function data. The analysis showed that whey protein supplementation had no effect on LM and muscle strength; nevertheless, a significant improvement was found in physical function (SMD = 0.561; 95% confidence interval [CIs]: 0.256, 0.865, n = 33), particularly gait speed (GS). On the contrary, whey protein supplementation significantly improved LM (SMD = 0.982; 95% CI: 0.228, 1.736; n = 11), appendicular lean mass and physical function (SMD = 1.211; 95% CI: 0.588, 1.834; n = 16), and GS in sarcopenic/frail older adults. By contrast, co-supplementation with vitamin D enhanced LM gains (SMD =0.993; 95% CI: 0.112, 1.874; n = 11), muscle strength (SMD =2.005; 95% CI: 0.975, 3.035; n = 11), and physical function (SMD = 3.038; 95% CI: 2.196, 3.879; n = 18) significantly. Muscle strength and physical function improvements after whey protein supplementation plus vitamin D were observed without resistance exercise (RE) and short study duration subgroups. Moreover, the combination of whey protein and vitamin D with RE did not enhance the effect of RE. Whey protein supplementation improved LM and function in sarcopenic/frail older adults but had no positive effect in healthy older persons. By contrast, our meta-analysis showed that co-supplementation with whey protein and vitamin D is effective, particularly in healthy older adults, which is likely owing, we propose, to the correction of vitamin D insufficiency or deficiency. The trial was registered at https://inplasy.com as INPLASY202240167.

Review question / Objective: Study Design A. Only systematic reviews and metaanalyses are considered.B. No narrative reviews or scoping reviews are considered.Participants Does the study involve adults aged ≥18 years?Groups that may be covered: A. Healthy older

BACKGROUND & AIMS:Diabetes mellitus (DM) and cardiovascular disease (CVD) are among the biggest causes of death and health expenses worldwide. A higher dietary acid load (DAL) is associated with chronic low-grade metabolic acidosis, and may increase the risk of insulin resistance (IR), DM, hypertension, and CVD mortality. However, the association between DAL and IR still lacks population-based studies to confirm laboratory findings. METHODS:This is a population-based observational study including a sample of 545 individuals aged 25-64 years from Florianópolis (Southern Brazil) who participated in the EpiFloripa cohort study. All diet variables were obtained through two 24-h Food Recalls adjusted to obtain an estimate of habitual food consumption. DAL was measured by Potential Renal Acid Load (PRAL) and Net Endogenous Acid production (NEAP). Fasting blood samples were obtained from all participants. The primary outcome was IR, which was estimated by HOMA-IR. Secondary outcomes included HOMA-β, glycosylated hemoglobin, and fasting blood glucose and insulin. Multiple linear regression models adjusted for sociodemographics, lifestyle, and clinical variables were used for analysis, with exposure and outcome variables standardized as Z-scores to allow comparability of the results. RESULT:The mean PRAL and NEAP in the sample were 16.9 ± 4.8 and 66.1 ± 7.1 mEq/day, respectively. The average HOMA-IR score was 2.4 ± 1.6. In adjusted analyses, PRAL was positively associated with HOMA-IR, fasting insulin, and fasting blood glucose (p-value <0.05 in all cases), but not with HOMA-β or glycated hemoglobin. NEAP also showed a direct-trend relationship with HOMA-IR and fasting insulin, but not with fasting blood glucose or the other outcomes. The strongest association was between PRAL and HOMA-IR (β, 0.20; 95% CI, 0.06-0.35). CONCLUSIONS:A higher DAL was consistently associated with higher IR and insulin levels but not with other glycaemic parameters. Apparently, β-pancreatic cells function is not affected by DAL in this population. This is the first study that describes the DAL in a population-based sample of adults in Latin America and in a middle-income country population. Further longitudinal and interventional studies are required to establish a better causal effect between DAL and IR.

Skeletal muscle plays a critical role in physical function and metabolic health. Muscle is a highly adaptable tissue that responds to resistance exercise (RE; loading) by hypertrophying, or during muscle disuse, RE mitigates muscle loss. Resistance exercise training (RET)-induced skeletal muscle hypertrophy is a product of external (e.g., RE programming, diet, some supplements) and internal variables (e.g., mechanotransduction, ribosomes, gene expression, satellite cells activity). RE is undeniably the most potent nonpharmacological external variable to stimulate the activation/suppression of internal variables linked to muscular hypertrophy or countering disuse-induced muscle loss. Here, we posit that despite considerable research on the impact of external variables on RET and hypertrophy, internal variables (i.e., inherent skeletal muscle biology) are dominant in regulating the extent of hypertrophy in response to external stimuli. Thus, identifying the key internal skeletal muscle-derived variables that mediate the translation of external RE variables will be pivotal to determining the most effective strategies for skeletal muscle hypertrophy in healthy persons. Such work will aid in enhancing function in clinical populations, slowing functional decline, and promoting physical mobility. We provide up-to-date, evidence-based perspectives of the mechanisms regulating RET-induced skeletal muscle hypertrophy.

The compound β-hydroxy-β-methyl butyrate (HMB) is proposed to increase or mitigate the loss of skeletal muscle and improve muscle function. We undertook a review of systematic reviews of HMB supplementation to promote gains or mitigate muscle loss in ageing and clinical populations. Following PRISMA guidelines, we searched for systematic reviews reporting the effect of HMB in our target populations. Dual-energy X-ray absorptiometry (DXA) measured lean soft-tissue mass (LSTM) was accepted as a proxy for muscle. We identified 15 systematic reviews that met our inclusion criteria, which were independently evaluated. The methodological quality of the reviews was assessed using A Measurement Tool to Assess Systematic Reviews (AMSTAR), and standardized effectiveness statements were generated. Five of 15 studies found some evidence that HMB augmented LSTM; the remaining 10 studies reported some evidence favouring no difference (6/10 studies) or insufficient evidence to determine an effect (4/10 studies). Of the 12 studies that evaluated strength, 4/12 found some evidence, 5/12 found some evidence of no effect with one article finding some evidence in favour of patients in peri-hospitalized and no evidence for those that are community-dwelling, 4/12 had insufficient evidence to determine an effect, and 1/12 had insufficient evidence. No]study reported a positive effect of HMB on physical function; however, 2/10 studies found some evidence favouring no effect, and 7/10 studies reported insufficient evidence to determine an effect. The effectiveness of HMB supplementation in augmenting LSTM was heterogeneous, with most reviews finding no effect or inconclusive evidence to determine an effect. Most reviews concluded that HMB supplementation did not affect strength outcome measures or studies were inconclusive. The current evidence is insufficient to assess the impact of HMB supplementation on functional outcome measures. Our analysis shows minor, inconsistent support for HMB as part of an oral nutritional supplement or as a stand-alone supplement (or combined with other amino acids) to increase or promote retention of LSTM, improve strength, and no evidence that it improves physical function in older persons or clinical populations.

Review question / Objective: Does a whey protein supplementation maintain or increase muscle mass, strength, and function in healthy older adults? Condition being studied: Sarcopenia is a state of age-related progressive loss of muscle mass and function. This syndrome is considered an undesirable consequence of aging, contributing to various negative health outcomes including increased risk of chronic metabolic diseases, physical disabilities, falls and fractures, reduced independence, frailty, and hospitalization. Due to population ageing in recent decades, the prevalence of sarcopenia has been growing considerably worldwide. In this context, sarcopenia has globally affected more than 29% of community-dwelling and 14-33% of institutionalized older adult populations. Thus, sarcopenia has been considered to be an important health concern. Indeed, it has been estimated that 500 million older adults would suffer from sarcopenia by 2050.

Abstract We performed a systematic review, meta‐analysis, and meta‐regression to determine if increasing daily protein ingestion contributes to gaining lean body mass (LBM), muscle strength, and physical/functional test performance in healthy subjects. A protocol for the present study was registered (PROSPERO, CRD42020159001), and a systematic search of Medline, Embase, CINAHL, and Web of Sciences databases was undertaken. Only randomized controlled trials (RCT) where participants increased their daily protein intake and were healthy and non‐obese adults were included. Research questions focused on the main effects on the outcomes of interest and subgroup analysis, splitting the studies by participation in a resistance exercise (RE), age (<65 or ≥65 years old), and levels of daily protein ingestion. Three‐level random‐effects meta‐analyses and meta‐regressions were conducted on data from 74 RCT. Most of the selected studies tested the effects of additional protein ingestion during RE training. The evidence suggests that increasing daily protein ingestion may enhance gains in LBM in studies enrolling subjects in RE (SMD [standardized mean difference] = 0.22, 95% CI [95% confidence interval] 0.14:0.30, P < 0.01, 62 studies, moderate level of evidence). The effect on LBM was significant in subjects ≥65 years old ingesting 1.2–1.59 g of protein/kg/day and for younger subjects (<65 years old) ingesting ≥1.6 g of protein/kg/day submitted to RE. Lower‐body strength gain was slightly higher by additional protein ingestion at ≥1.6 g of protein/kg/day during RE training (SMD = 0.40, 95% CI 0.09:0.35, P < 0.01, 19 studies, low level of evidence). Bench press strength is slightly increased by ingesting more protein in <65 years old subjects during RE training (SMD = 0.18, 95% CI 0.03:0.33, P = 0.01, 32 studies, low level of evidence). The effects of ingesting more protein are unclear when assessing handgrip strength and only marginal for performance in physical function tests. In conclusion, increasing daily protein ingestion results in small additional gains in LBM and lower body muscle strength gains in healthy adults enrolled in resistance exercise training. There is a slight effect on bench press strength and minimal effect performance in physical function tests. The effect on handgrip strength is unclear.

Acute mental stress (AMS) increases heart rate (HR) and blood pressure. Since obesity can impair the cardiovascular reactivity to AMS, a better understanding of the mechanisms involved in this response is needed. We aimed to evaluate the cardiovascular reactivity to AMS in young men with normal or excess body fat. We also assessed the association between cardiovascular reactivity to AMS and cardiovascular risk factors, including autonomic modulation, carotid artery distensibility, physical activity levels, and sleep efficiency. Sixty-six young men (26.1 +/- 4.1 years old) underwent anthropometric and body fat assessment (dual-energy X-ray absorptiometry) and had right-carotid artery ultrasonography. Accelerometers assessed physical activity levels and sleep efficiency. AMS was induced through the Stroop color-word test while blood pressure, HR, and cardiac interval were measured. Analyses were performed in Normal and Excess fat groups divided by fat mass index (FMI). Continuous data was used for multiple linear regression analyses. An interaction between FMI and time for HR reactivity was observed. Cardiac interval variability analysis showed that only participants with normal fat displayed parasympathetic withdrawal during AMS (P < 0.05). Multiple linear regression analysis supported the role of adiposity and autonomic modulation in the HR reactivity to AMS and showed involvement of carotid distensibility and sleep efficiency (P < 0.05). Carotid distensibility was the only predictor for blood pressure reactivity (P < 0.05). Physical activity was not associated with AMS's cardiovascular reactivity. We conclude that increased adiposity is associated with reduced HR reactivity to AMS, which is possibly linked to an impaired parasympathetic withdrawal. Carotid distension and sleep efficiency seem to contribute to this response.

This systematic review, meta-analysis, and meta-regression aimed to determine if increasing daily protein ingestion contributes to gaining lean mass (LM), muscle strength, and physical/functional test performance in healthy persons. The present review was registered on PROSPERO - CRD42020159001. A systematic search in Medline, Embase, CINAHL, and Web of Sciences databases was undertaken. Randomized controlled trials (RCT) including healthy and non-obese adult participants increasing daily protein intake were selected. Subgroup analysis, splitting the studies by participation in resistance exercise training (RE), age (< 65 or ≥ 65 y), and daily protein ingestion were also performed. 74 RCT fit our inclusion criteria. The age range of the participants was 19 to 85 y, and study protocols in the trials lasted from 6 to 108 wks (76% of the studies between 8 and 12 wks). In ∼80% of the studies, baseline protein ingestion was at least 1.2 g of protein/kg/d. Increasing daily protein ingestion may lead to small gains in LM in subjects enrolled in RE (SMD [standardized mean difference] = 0.22, CI95% [95% confidence interval] 0.14:0.30, P < 0.01, 62 studies, moderate level of evidence). Also, ≥ 65 y subjects ingesting 1.2–1.59 g of protein/kg/d and younger subjects (< 65 y) increasing their ingestion to ≥ 1.6 g of protein/kg/d during RE showed a higher LM gain. Lower-body strength gain was slightly higher at ≥ 1.6 g of protein/kg/d during RE (SMD = 0.40, CI95% 0.09:0.35, P < 0.01, 19 studies, low level of evidence). Bench press strength was slightly increased by ingesting more protein in < 65 y subjects during RE (SMD = 0.18, CI95% 0.03:0.33, P = 0.01, 32 studies, low level of evidence). Effects on handgrip strength are unclear and only marginal for performance in physical function tests. The number of studies increasing daily protein ingestion alone was too low (n = 6) to conduct a meta-analysis. The current evidence shows that increasing protein ingestion by consuming supplements or food, resulted in small additional gain in LM, and lower body muscle strength in healthy adults enrolled in RE. Effects on bench press strength and performance in physical function tests are minimal. The effect on handgrip strength was unclear. This research received a grant from the International Life Science Institute (Europe) and CNPq.

BACKGROUND The energy-adjusted Dietary Inflammatory Index (E-DIITM ) has been associated with high body mass index and markers of chronic diseases. Also, pro-inflammatory diets with high E-DII have been positively associated with metabolic disturbances such as glucose intolerance and type II diabetes mellitus. However, it is unclear if E-DII scores are positively associated with body fat percentage and visceral fat per se. This cross-sectional study aimed to evaluate whether the E-DII is associated with body fat content and metabolic health indicators in lean and obese young men. METHODS The study was conducted on 59 participants, without comorbidities, not using tobacco, medication, and nutritional supplements. Dietary data were obtained by 3-days food records to calculate E-DII scores based on 28 food parameters. Body composition was assessed by dual x-ray absorptiometry (DXA). Blood samples were taken to measure fasting glucose, insulin, triacylglycerols, total cholesterol, and low- and high-density lipoprotein cholesterol (LDL-C and HDL-C). An oral glucose tolerance test also was performed. Associations were determined by mixed-effects linear regression. RESULTS E-DII scores ranged from -3.48 to +3.10. Energy intake was similar across E-DII tertiles. After adjusting for covariates, the highest E-DII tertile was associated with increased body fat, visceral adipose tissue, and waist circumference. There was no association between E-DII scores and lipid or glycemic parameters. CONCLUSION In young participants, a dietary pattern with a higher E-DII (i.e., pro-inflammatory) score was associated with high body fat and markers of central adiposity assessed by DXA, regardless of body mass. This article is protected by copyright. All rights reserved.

ABSTRACT It is well established that exercise is associated with a reduced risk of several chronic diseases. Currently, aerobic training (AT) receives primary attention in physical activity guidelines with a recommendation for ~150 min of moderate-to-vigorous AT weekly. In most physical activity guidelines, resistance training (RT) is termed a beneficial activity, with a recommendation to engage in strengthening activities twice weekly. However, we propose that the health benefits of RT are underappreciated. There is evidence, established and emerging, that RT can, in many respects, elicit similar health benefits to AT. When combined, AT and RT may yield ostensibly optimal health benefits versus performing either exercise exclusively. We discuss the health benefits of engaging in RT, including healthy aging, improved mobility, cognitive function, cancer survivorship, and metabolic health in persons with obesity and type 2 diabetes—all of which can influence morbidity and mortality. Many of the health benefits of RT can be achieved by lifting lighter loads to volitional failure, highlighting that the benefits of RT do not necessarily require lifting heavier weights. Accumulating evidence also shows a lower mortality risk in those who regularly perform RT. To optimize health, especially with aging, RT should be emphasized in physical activity guidelines in addition to AT.

Background: The coronavirus disease (COVID-19) pandemic has promoted changes in lifestyle behaviors, such as food consumption, sleep, and physical activity (PA). Few longitudinal studies have investigated these changes in young adults. Aim: This study aimed to assess lifestyle behaviors before and during the COVID-19 pandemic in young adult males. Methods: 50 young adult males (18–35 years) recruited by posters and social media in Florianopolis, Brazil, provided data on food consumption, PA, and sleep in 2018–2019 (baseline) and during the pandemic in 2020 (follow-up). PA and sleep variables were assessed through self-reported questionnaires. Food records were used to evaluate food consumption. Weight was measured using Bioelectrical impedance analysis at baseline and using self-reported at follow-up. Multilevel linear regression models and generalized linear multilevel were used to test differences between baseline and follow-up. Results: The findings indicated significant changes at follow-up, compared to baseline. Decreased consumption of total fat (β = −13.32, 95% CI (−22.45; −4.18), p < 0.01), sodium (β = −1330.72, 95% CI (−1790.63; −870.82), p < 0.01), cholesterol (β = −212.99, 95% CI (−269.8; −156.18), p < 0.01), total sugars (β = −65.12, 95% CI (−80.94; −49.29), p < 0.01), alcohol, and sugar-sweetened beverage were observed. Despite that, a slight increase in weight was also observed (80.70 ± 16.37 kg vs. 82.99 ± 15.42 kg, p = 0.000748). Sleep duration increased (β = 0.7596, 95% CI (0.41; 1.11), p < 0.01), and occupational PA decreased (β = −1168.1, 95% CI (−1422.33; −913.83), p < 0.01), while domestic (β = 394.04, 95%CI (114.68; 673.39, p < 0.01)) and leisure PA (β = 499.91, 95% CI (245.28; 754.53), p < 0.01) increased. Conclusion: Our results suggest that social distancing policies positively impacted eating habits, sleep, and PA patterns. These changes are possibly linked to increased awareness of the need for a healthy lifestyle.

Decreased skeletal muscle contractile activity (disuse) or unloading leads to muscle mass loss, also known as muscle atrophy. The balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB) is the primary determinant of skeletal muscle mass. A reduced mechanical load on skeletal muscle is one of the main external factors leading to muscle atrophy. However, endocrine and inflammatory factors can act synergistically in catabolic states, amplifying the atrophy process and accelerating its progression. In addition, older individuals display aging-induced anabolic resistance, which can predispose this population to more pronounced effects when exposed to periods of reduced physical activity or mechanical unloading. Different cellular mechanisms contribute to the regulation of muscle protein balance during skeletal muscle atrophy. This review summarizes the effects of muscle disuse on muscle protein balance and the molecular mechanisms involved in muscle atrophy in the absence or presence of disease. Finally, a discussion of the current literature describing efficient strategies to prevent or improve the recovery from muscle atrophy is also presented.

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH(2)O(2)) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH(2)O(2), protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

Leucine is a critical amino acid stimulating myofibrillar protein synthesis (MyoPS). The consumption of higher leucine-containing drinks stimulates MyoPS, but we know less about higher leucine solid foods. Here, we examined the effect of short-term resistance exercise training (STRT) combined with supplementation of a protein and leucine-enriched bar, com-pared with STRT alone, on integrated (%/day) rates of MyoPS and anabolic protein signaling. In a nonblinded, randomized crossover trial, eight young adults performed four sessions of STRT without or while consuming the study bar (STRT+Leu, 16 g of protein containing similar to 3 g of leucine) for two 4-day phases, separated by 2 days nonexercise (Rest) washout. In combination with serial muscle biopsies, deuterated water permitted the measurement of MyoPS and protein signaling phosphorylation. MyoPS during STRT (1.43 +/- 0.06%/day) and STRT+Leu (1.53 +/- 0.06%/day) were greater than Rest (1.31 +/- 0.05%/day), and MyoPS during STRT+Leu (1.53 +/- 0.06%/day) was greater than STRT alone (1.43 +/- 0.06%/day). STRT+Leu increased the ratio of phospho-rylated to total mechanistic target of rapamycin and 4EBP1 compared to Rest. Engaging in STRT increased integrated MyoPS and protein signaling in young adults and was enhanced with increased protein intake derived from a leucine-enriched protein bar. This study was registered at clinicaltrials.gov as NCT03796897.