Adequate protein intake is essential for the maintenance of whole-body protein mass. Different methodological approaches are used to substantiate the evidence for the current protein recommendations, and it is continuously debated whether older adults require more protein to counteract the age-dependent loss of muscle mass, sarcopenia. Thus, the purpose of this critical narrative review is to outline and discuss differences in the approaches and methodologies assessing the protein requirements and, hence, resulting in controversies in current protein recommendations for healthy older adults. Through a literature search, this narrative review first summarises the historical development of the Food and Agriculture Organization/World Health Organization/United Nations University setting of protein requirements and recommendations for healthy older adults. Hereafter, we describe the various types of studies (epidemiological studies and protein turnover kinetic measurements) and applied methodological approaches founding the basis and the different recommendations with focus on healthy older adults. Finally, we discuss important factors to be considered in future studies to obtain evidence for international agreement on protein requirements and recommendations for healthy older adults. We conclude by proposing future directions to determine ‘true’ protein requirements and recommendations for healthy older adults.

Unavoidable periods of disuse lead to muscle atrophy and functional decline. Preventing such declines can reduce the risk of re-injury and improve recovery of normal physiological functioning. We aimed to determine the effectiveness of high-dose leucine supplementation on muscle morphology and strength during 7 d of unilateral lower-limb immobilization, and the role of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis in disuse atrophy. Sixteen healthy males (mean ± SEM age: 23 ± 1 y) underwent 7 d of unilateral lower-limb immobilization, with thrice-daily leucine (LEU; n = 8) or placebo (PLA; n = 8) supplementation (15 g/d). Before and after immobilization, muscle strength and compartmental tissue composition were assessed. A primed continuous infusion of l-[ring-¹³C₆]-phenylalanine with serial muscle biopsies was used to determine postabsorptive and postprandial (20 g milk protein) MyoPS and MitoPS, fiber morphology, markers of protein turnover, and mitochondrial function between the control leg (CTL) and the immobilized leg (IMB). Leg fat-free mass was reduced in IMB (mean ± SEM: −3.6% ± 0.5%; P = 0.030) but not CTL with no difference between supplementation groups. Isometric knee extensor strength declined to a greater extent in IMB (−27.9% ± 4.4%) than in CTL (−14.3% ± 4.4%; P = 0.043) with no difference between groups. In response to 20 g milk protein, postprandial MyoPS rates were significantly lower in IMB than in CTL (−22% ± 4%; P < 0.01) in both LEU and PLA. Postabsorptive MyoPS rates did not differ between legs or groups. Postabsorptive MitoPS rates were significantly lower in IMB than in CTL (−14% ± 5%; P < 0.01) and postprandial MitoPS rates significantly declined in response to 20 g milk protein ingestion (CTL: −10% ± 8%; IMB: −15% ± 10%; P = 0.039), with no differences between legs or groups. There were no significant differences in measures of mitochondrial respiration between legs, but peroxisome proliferator–activated receptor γ coactivator 1-α and oxidative phosphorylation complex II and III were significantly lower in IMB than in CTL (P < 0.05), with no differences between groups. High-dose leucine supplementation (15 g/d) does not appear to attenuate any functional declines associated with 7 d of limb immobilization in young, healthy males. This trial was registered at clinicaltrials.gov as NCT03762278.

Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.

Skeletal muscle protein turnover plays a crucial role in controlling muscle mass and protein quality control, including sarcomeric (structural and contractile) proteins. Protein turnover is a dynamic and continual process of protein synthesis and degradation. The ubiquitin proteasome system (UPS) is a key degradative system for protein degradation and protein quality control in skeletal muscle. UPS-mediated protein quality control is known to be impaired in aging and diseases. Exercise is a well-recognized, nonpharmacological approach to promote muscle protein turnover rates. Over the past decades, we have acquired substantial knowledge of molecular mechanisms of muscle protein synthesis after exercise. However, there have been considerable gaps in the mechanisms of how muscle protein degradation is regulated at the molecular level. The main challenge to understand muscle protein degradation is due in part to the lack of solid stable isotope tracer methodology to measure muscle protein degradation rate. Understanding the mechanisms of UPS with the concomitant measurement of protein degradation rate in skeletal muscle will help identify novel therapeutic strategies to ameliorate impaired protein turnover and protein quality control in aging and diseases. Thus, the goal of this present review was to highlight how recent advances in the field may help improve our understanding of exercise-mediated protein degradation. We discuss 1) the emerging roles of protein phosphorylation and ubiquitylation modifications in regulating proteasome-mediated protein degradation after exercise and 2) methodological advances to measure in vivo myofibrillar protein degradation rate using stable isotope tracer methods.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D₂O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

Dietary protein has a pivotal role in muscle mass maintenance with advancing age. However, an optimal dose and distribution of protein intake across the day as well as the interaction with energy intake for the maintenance of muscle mass and physical function in healthy older adults remain to be fully elucidated. The purpose of this study was to examine the association between muscle mass, strength, and physical function, and the total amount and distribution of protein and energy intake across the day in healthy older individuals. The research question was addressed in a cross-sectional study including 184 Danish men and woman (age: 70.2 ± 3.9 years, body mass: 74.9 ± 12.1 kg, Body Mass Index (BMI): 25.4 ± 3.7 kg/m²) where a 3-day dietary registration, muscle mass, strength, and functional measurements were collected. We found that neither daily total protein intake nor distribution throughout the day were associated with muscle mass, strength, or physical function. Consequently, we do not provide an incentive for healthy older Danish individuals who already adhere to the current internationally accepted recommended dietary protein intake (0.83 g/kg/day) to change dietary protein intake or its distribution pattern throughout the day.