Nutrients 2019, 11, 727 2 of 12
postprandial anabolic response in skeletal muscle occurs through the stimulation of proteinsynthesis initiated by increased essential amino acid bioavailability, whereas the decrease in proteolysis mainly occurs through the stimulation of insulin secretion in response to feeding [ 2 , 3 ]. Although decreased physical activity has been identified as a cause, muscle atrophy has also been explained by the presence of anabolic resistance following food intake in several physio-pathological catabolic states as diverse as aging, cancer, and disuse [ 4 – 10 ]. In each case, increasing dietary protein intake above the current recommended dietary allowances (RDA at 0.8 g/kg/day) [ 11 ] has been proposed to overcome the anabolic resistance observed. Among dietary proteins, rapidly digested and leucine-rich proteins (i.e., whey) have been shown to be the most efficient for the acute stimulation of muscleproteinsynthesis during aging, disuse or glucocorticoid treatment when compared to casein, a protein with lower leucine content and whose digestion rate is slower [ 9 , 12 – 14 ]. However, when given in the long-term, whey proteins alone do not appear to be an optimal nutritional strategy to prevent or slow down muscle wasting during aging [ 15 , 16 ] or catabolic states [ 10 , 17 – 19 ]. This could be explained by the nature and intensity of the catabolic state but also by the fact that the digestion of whey may be too rapid during a catabolic situation to sustain the anabolic postprandial amino acid requirement necessary to elicit an optimal anabolic response [ 20 ]. Indeed, the stimulation of muscleproteinsynthesis has a defined and limited duration during the postprandial period [ 21 , 22 ]. Atherton et al. showed that whey ingestion is only able to stimulate muscleproteinsynthesis for 2 h [ 21 ]. The leucine content of a complete meal drives peak activation but not the duration of skeletal muscleproteinsynthesis [ 23
activities of intracellular kinases linked to the translation of proteins such as mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 (p70S6K) kinases (Kimball et al. 1999; Anthony et al. 2000b; Dardevet et al. 2000). We recently demonstrated in vitro that proteinsynthesis in old rat muscles becomes resistant to the stimulatory effect of leucine at its physiological concentration range (Dardevet et al. 2000). However, when leucine concentration was increased greatly above its postprandial level, proteinsynthesis was stimulated normally (Dardevet et al. 2002; Rieu et al. 2003) and the inhibition of muscleprotein breakdown was restored in old rats (Combaret et al. 2005). Based on our observations, dietary leucine supplementation may represent a useful nutritional tool for the maintenance of muscle mass and the prevention of sarcopenia in the elderly. To our knowledge, the beneficial effect of a specific leucine supplementation in aged humans has only been shown by Katsanos et al. (2006) in combination with a bolus of EAAs. Whether a specific leucine effect on muscleproteinsynthesis can be obtained with leucine-supplemented meals under normal postprandial conditions (i.e. in the presence of carbohydrates and lipids) remains to be demonstrated. Indeed, Volpi et al. (2000) showed that the response of muscleprotein anabolism to a large amino acid intake was blunted in combination with other nutrients (especially glucose) in the elderly. The aim of the present study was to evaluate the effect of complete meals (containing protein, carbohydrates and lipids) enriched or not with leucine on whole body protein metabolism and muscleproteinsynthesis in elderly volunteers. Methods
† These authors contributed equally to this study.
Received: 27 March 2020; Accepted: 26 May 2020; Published: 29 May 2020
Abstract: The mechanisms that are responsible for sarcopenia are numerous, but the altered muscleprotein anabolic response to food intake that appears with advancing age plays an important role. Dietary protein quality needs to be optimized to counter this phenomenon. Blending different plant proteins is expected to compensate for the lower anabolic capacity of plant-based when compared to animal-based protein sources. The objective of this work was to evaluate the nutritional value of pasta products that were made from a mix of wheat semolina and faba bean, lentil, or split pea flour, and to assess their effect on protein metabolism as compared to dietary milk proteins in old rats. Forty-three old rats have consumed for six weeks isoproteic and isocaloric diets containing wheat pasta enriched with 62% to 79% legume protein (depending on the type) or milk proteins, i.e., casein or soluble milk proteins (SMP). The protein digestibility of casein and SMP was 5% to 14% higher than legume-enriched pasta. The net protein utilization and skeletal muscleproteinsynthesis rate were equivalent either in rats fed legume-enriched pasta diets or those fed casein diet, but lower than in rats fed SMP diet. After legume-enriched pasta intake, muscle mass, and protein accretion were in the same range as in the casein and SMP groups. Mixed wheat-legume pasta could be a nutritional strategy for enhancing the protein content and improving the protein quality, i.e., amino acid profile, of this staple food that is more adequate for maintaining muscle mass, especially for older individuals. Keywords: sarcopenia; skeletal muscle; legume-enriched pasta; faba bean; lentil; split pea; protein quality; muscleproteinsynthesis rate
impact on skeletal muscleproteinsynthesis remains
In the present study, muscleproteinsynthesis was increased with WP dietary protein in AL-fed old rats. Furthermore, although dietary restrictions decreased muscleprotein syn- thesis, regardless of the ER or PER conditions, WP rats dis- played a greater muscleproteinsynthesis rate than their CAS counterparts. Overall, this effect may participate in preventing muscle loss in PER-WP and ER-WP old rats. In the AL-WP old rats, the unchanged muscle mass compared with AL-CAS rats despite the higher proteinsynthesis rate would suggest that muscle proteolysis was also enhanced in this group, resulting in higher muscleprotein turnover. This result contrasts with previous whole-body studies supporting the idea that ‘fast’
leucine, activate the mTORC1 pathway and consequently stim- ulate MPS (12).
In the present study, we show that CIT supplementation in malnourished aged rats is able to stimulate the mTORC1 pathway in the skeletal muscle. This experimental result is in line with those of others using a model of short fasting (22) but seems to conflict with those of Churchward-Venne et al. (7). These authors did not observe any effect of CIT on muscleproteinsynthesis or mTORC1 activation in elderly men. This discrepancy could be linked to marked differences between our work and the design of this study. First, Churchward-Venne et al. (7) compared the effect of a very high dose of protein (45 g of whey protein) or high dose of protein (15 g of protein) plus CIT. In these conditions, they were not exploring the effect of CIT but rather, how replacement of some protein by CIT would have the same effect (not the same question). Second, consid- ering the effect on the mTORC1 pathway, they did not observe any effect of CIT (in combination with whey protein) between the two groups studied, but the authors did not evaluate mTORC1 activation in basal conditions, which does not allow any conclusion to be drawn about the possible effect of CIT. However, in vivo, the anabolic effect of CIT on muscle could be indirect and mediated by hormonal change, such as stimu- lation of insulin secretion. Insulin is known to stimulate MPS through activation of the PI3K/Akt pathway. However, this is unlikely because no effect of CIT was observed on insulin secretion in either animal (22) or human studies (20, 29). This hypothesis is supported at the transductional level, since we observed no CIT-related activation of Akt (the main insulin target). Taken together, these data show that the anabolic action of CIT is independent of insulin and the Akt pathway. Interestingly, we observed a positive correlation between plasma CIT concentration (but not muscle CIT concentration) and S6K1 phosphorylation. Similar results were obtained in a previous study (34) in a model of short bowel syndrome rats in
2.2. In vivo muscleproteinsynthesis
MPS was measured with the SUnSET method as previously described by Goodman et al.  and previously used by us [22,24] . The SUnSET method is an alternative method to determine MPS compared to radioactive isotope or stable isotope tracers. Goodman et al.  validated the SUnSET method with a 3 H-phenylalanine ﬂooding method in ex vivo plantaris muscle from mice that had received synergist ablation (SA). The results showed that the SUn- SET technique was indistinguishable from a standard radioactive- based or a standard stable isotope incorporation technique in detecting SA-induced increases in proteinsynthesis. In addition, in our previous study  , proteinsynthesis was measured in myo- tubes after a 50 min incubation with L-[1- 13 C]valine by measuring tracer enrichment, or a 30 min incubation with puromycin by quantifying incorporation of puromycin into peptides. Results of both methods were comparable. Furthermore, we used this tech- nique successfully in a previous study were similar dosages of protein showed an anabolic response in adult mice  . This proves the reliability of the SUnSET method. This method also made it possible to determine proteinsynthesis rate and activation of key signalling pathways involved in this process using the same experimental samples.
et al. 1994; Oosterveer et al. 2009) and an increase in
lipid uptake by skeletal muscles for oxidation (Slawik & Vidal-Puig, 2007).
In conclusion, muscle and adipose tissue mass, intra- myocellular lipids (IMCL) and muscleproteinsynthesis were differently affected according to the stage of obesity development. IMCL and muscleproteinsynthesis were also differently affected depending on muscle typology. The second phase of obesity development was associated with a reduction of adipose tissue expandability and an increase of IMCL in glycolytic muscle, which is concomitant with lower proteinsynthesis stimulation firstly observed with the high-fat, high-sucrose diet. These data suggest that IMCL accumulation is deleterious for the incorporation of amino acids in newly synthetized skeletal muscle proteins and could contribute to the loss of skeletal muscle mass and quality. Moreover, this process, especially if co-morbidities of obesity occur, could lead ultimately to sarcopenic obesity, which is defined by a combination of excess weight and reduced muscle mass or strength (Zamboni et al. 2008). It could be inter- esting to see if strategies used to treat obesity, for example caloric restriction and exercise, may reduce muscle lipid infiltration and change the specific protein metabolism modifications observed in skeletal muscle during obesity.
In conclusion, citrulline supplementation in the old malnour- ished rats increases protein content of the muscle by stimulat- ing proteinsynthesis. Whether this effect is transposable to humans and whether this strategy can improve the clinical outcome of elderly malnourished patients requires further study. Also, further work is needed to determine the mecha- nisms (direct or indirect) involved in citrulline action. Also, there is a splanchnic sequestration of amino acids in a number of pathological situations (including trauma, cancer, and type 2 diabetes). Although the underlying mechanisms here are cer- tainly different from those encountered with advanced age, evaluating the effects of citrulline supplementation in these various situations is of interest because they are all character- ized by impaired muscleproteinsynthesis.
From other perspectives, a person’s poor dentition can limit chewing capacity and protein availability (50), as can under- or over-cooking of protein foods.(51)
Protein intake pattern
While terminology used to describe patterns of protein intake varies, intake patterns in research studies were to “spread” protein evenly over 4 meals or to deliver protein mostly as a large “pulse” in a single meal. Results of several studies suggest that the pulse protein feeding pattern may be useful to improve feeding-induced stimulation of proteinsynthesis in older adults.(52–54) These results are seemingly contradictory to those suggesting that 4 doses of 20 gram of protein across 12 hours is the optimal pattern.(55) It is not clear whether the discrepancy between these studies is due to age differences, in inclusion of exercise, or most likely, the fact that 20 gram protein is not enough to maximally stimulate muscleproteinsynthesis in older adults. Further studies are needed to clarify optimal patterns of protein intake for older adults, and such studies must include proteinsynthesis as well as improvements in muscle strength and performance as outcome measures.
Competing Interests: The authors have declared that no competing interests exist. * E-mail: firstname.lastname@example.org
Loss of skeletal muscle mass occurs during aging (sarcopenia), disease (cachexia), or inactivity (atrophy) and results from an imbalance between the rate of muscleproteinsynthesis and degradation . In a previous study, we identified SREBP-1 transcription factors as regulators of muscle mass, showing that increasing SREBP-1 nuclear content induces both in vitro and in vivo muscle cell atrophy . The Sterol Regulatory Element Binding Proteins (SREBP) transcription factors belong to the basic helix-loop-helix leucine zipper family of DNA binding proteins . The three isoforms are encoded by two distinct genes, Srebf1 and Srebf2, and vary in structure, regulation, and functions: SREBP-1a and SREBP-1c proteins are key actors of the regulation of genes related to lipid metabolism, whereas SREBP-2 has been more closely associated to cholesterol synthesis and accumulation .
The DUX4 and PITX1 proteins half-lifes are regulated by the ubiquitin-proteasome pathway
Our data indicate that, similar to other transcription factors, the DUX4 protein stability appears highly regulated, likely in relation to a role in early development . We found that DUX4 was degraded by the ubiquitin-proteasome pathway (UPP), likely targeting its carboxyl-ter- minal domain, which contains sequences with destabilization proba- bility (Fig. S5B). Because it is a very potent transcriptional activator , very small amounts of DUX4 could be sufficient to initiate the deregulation cascade. We should also mention that although the pro- teasome usually completely degrades its substrates into small pep- tides, in a few cases, its proteolytic activity yields biologically active protein fragments as described for several transcription factors (NF- kappa B, Spt23p and Mga2p) . In future studies, it will be interest- ing to evaluate whether such a proteasomal processing could also occur for members of the FSHD transcriptional cascade, leading to smaller fragments that might exert some biological activity. Finally, our results suggested that other degradation pathways could interfere with DUX4 stability. Indeed, the use of MG132 alone did not always sufficiently stabilize DUX4 to allow its codetection with the product of its PITX1 target gene. The dynamic expression model presented here, together with an asynchronous regulation of their half-life by the pro- teasome could explain why the DUX4 and PITX1 proteins were Fig. 6 DUX4 expression in facioscapulohumeral muscular dystrophy
During endosperm development in cereals, GSP syn- thesis is mainly controlled at a transcriptional level. The regulatory mechanisms of GSP gene expression in barley have been described as a network of cis-motifs and their interacting transcription factors (TFs) (Rubio-Somoza et al., 2006a,b; Moreno-Risueno et al., 2008). This network is conserved in other cereals and dicots, as reviewed by Verdier and Thompson (2008) and Xi and Zheng (2011). The bipartite endosperm box has been identified in the promoter of some hordein and LMW-GS genes (Ham- mond-Kosack et al., 1993; O~nate et al., 1999; Juhasz et al., 2011). It contains two distinct protein-binding sites, the GCN4-like motif (GLM, 5 0 -ATGAG/CTCAT-3 0 ) and the pro- lamin box (P-box, 5 0 -TGTAAAG-3 0 ), and it plays a key role in activating the expression of GSP genes. The GLM and the P-box are recognized by basic leucine zipper (bZIP) and DNA binding with one finger (DOF) TFs, respectively. The GLM is recognized by BLZ1 and BLZ2 in barley (Vice- nte-Carbajosa et al., 1998; O~nate et al., 1999), RISBZ1, REB and RITA-1 in rice (Izawa et al., 1994; Nakase et al., 1997; Onodera et al., 2001), Opaque-2 (O2), OHP1 and OHP2 in maize (Pysh et al., 1993; Zhang et al., 2015) and SPA in wheat (Albani et al., 1997). The P-box is bound by BPBF and SAD in barley and WPBF in wheat (Vicente-Car- bajosa et al., 1997; Mena et al., 1998; Diaz et al., 2005). Two additional cis-elements have been described in bar- ley: 5 0 -AACA/TA-3 0 binds GAMYB, a TF of the R2R3MYB family, and 5 0 -TATC/GATA-3 0 binds HvMCB1 and HvMYBS3, two regulatory proteins of the R1MYB family (Diaz et al., 2002; Rubio-Somoza et al., 2006a,b). Another important motif, the RY box (5 0 -CATGCATG-3 0 ), is recog- nized by FUSCA3, a B3-type TF in barley and wheat (Mor- eno-Risueno et al., 2008; Sun et al., 2017). Differences in the organization of regulatory cis-elements have been noted in wheat GSP promoters. The long endosperm box in the promoter of the LMW-GS gene GluD3 reported by Hammond-Kosack et al. (1993), which contains two copies of the endosperm box, is not present in all LMW- GS promoters (Juhasz et al., 2011). The HMW-GS gene promoter contains an atypical endosperm box where the P-box is associated with a G-like box able to bind bZIP proteins like O2 and SPA (Norre et al., 2002; Ravel et al., 2014). Recently a common framework of cis-regulation was found for all HMW-GS gene promoters (Ravel et al., 2014), based on a composite box made of the GATA and GLM motifs (named the GATA-GLM box). This box is
Fouling is a significant challenge for membrane filtration since it diminishes the separation capabilities and increases the cost for membrane cleaning and replacement. Fouling is caused by accumulation, deposition and adsorption of particles, colloids or biological molecules on the membrane surface (external fouling) or within membrane pores (internal fouling) . Researchers have focused on the surface modification of synthetic membranes using graft polymerization to reduce fouling [20–23]. However, only a few low fouling membranes have been discovered over the past 30 years. Previously, we described a high throughput platform (HTP) combined with a photo-grafting method that facilitated quick synthesis and screening of protein-resistant membrane surfaces from a library of commercial vinyl monomers [24–27]. Membrane surfaces grafted with hydroxyl, PEG, amine and zwitterionic monomers exhibited protein resistance. Moreover, based on these HTP results, the mechanism of non-fouling has also been investigated with the help of structure–property relationships . As the number of the commercially available vinyl monomers is limited, there are opportunities to expand this library approach, increase the variety of protein- resistant surfaces, and explore protein adhesion or fouling mechanisms.
Fig. 2. Microsporidian LeuRS synthetase lacks a proofreading domain and is naturally resistant to a drug from the benzoxaborole family. (A) Schematic structures of aminoacyl-tRNA synthetase LeuRS from different species, including several species of microsporidian parasites. The structures are color-coded by conservation: conserved segments are shown in blue ( >50% sequence similarity between E. cuniculi and S. cerevisiae), segments degenerated in microsporidia but conserved in other eukaryotes are in green, and hypervariable segments in microsporidian LeuRS are in red (sequence similarity <35% between E. cuniculi and T. hominis). The scale indicates LeuRS length (in amino acids), and the silhouette symbols indicate a best-studied host (human, monkfish, honey bee, and silkworm) for each microsporidian parasite. The figure illustrates that, unlike LeuRS from other eukaryotic species, microsporidian LeuRS synthetases have a massively truncated editing domain, suggesting that microsporidian LeuRS synthetases lacks the editing activity. (B) Aminoacyl-tRNA hydrolysis assay to measure hydrolysis of the misaminoacylated substrate, Ile-tRNA Leu , by microsporidian (E. cuniculi) or yeast (S. cerevisiae) LeuRS. The figure shows that unlike yeast LeuRS (used as a positive control), LeuRS from E. cuniculi is not capable of degrading the Ile-tRNA Leu , illustrating a lack of the editing activity. (C) Kinetics of Leu-tRNA Leu synthesis by microsporidian (E. cuniculi) and yeast (S. cerevisiae) LeuRS in the absence and in the presence of AN2690, a member of the benzoxaborole family that targets the editing domain of LeuRS synthetase. While both S. cerevisiae and E. cuniculi LeuRS synthetases are capable of pro- ducing Leu-tRNA Leu in the absence of AN2690, only microsporidian LeuRS synthetase remains active in the presence of the drug, illustrating the natural resistance of E. cuniculi LeuRS to benzoxaboroles.
The aim of the present study was to determine whether the addition of soluble fibre in the diet affected protein metabolism in the intestinal tissues, some visceral organs and in skeletal muscle. A diet supplemented with pectin (80 g/kg) was fed to young growing rats and the effect on organ mass and protein metabolism in liver, spleen, small and large intestines and gastrocnemius muscle was monitored and compared with the control group. Proteinsynthesis rates were determined by measuring [ 13 C]valine incorporation in tissue protein. In the pectin-fed rats compared with the controls, DM intake and body weight gain were reduced (9 and 20 %, respectively) as well as gastrocnemius muscle, liver and spleen weights (6, 14 and 11 %, respectively), but the intestinal tissues were increased (64 %). In the intestinal tissues all protein metabolism parameters (protein and RNA content, proteinsynthesis rate and translational efficiency) were increased in the pectin group. In liver the translational efficiency was also increased, whereas its protein and RNA contents were reduced in the pectin group. In gastrocnemius muscle, protein content, fractional and absol- ute proteinsynthesis rates and translational efficiency were lower in the pectin group. The stimulation of protein turnover in intestines and liver by soluble fibre such as pectins could be one of the factors that explain the decrease in muscle turnover and whole-body growth rate.
muscle defects and not subsequent to muscle
MAP6 KO mice show muscle function defects in vivo
Muscle function was further studied in the MAP6 KO mouse line. Due to the multiple behavioral defects of these mice, like an acute response to stress [ 28 – 30 ], the use of classical force measurement tests (treadmill run or grip tests) was not possible. Therefore, we used a non-invasive protocol involving transcutaneous electro- stimulation coupled to mechanical measurement and multimodal magnetic resonance (MR) acquisition to evaluate gastrocnemius muscle function in anesthetized animals [ 41 ]. Because the absence of MAP6 results in synaptic function alteration [ 32 , 47 ], muscle electrosti- mulation was performed by a direct depolarization of the plasma membrane, bypassing nerve stimulation. The muscle volume and the twitch tension developed during a 6-min fatiguing bout of exercise were measured in 8 months old male mice (Fig. 3 a, b and Table 2 ).
Aminoacyl-tRNAs are used as a source of activated amino acids for non-ribosomal synthesis of various biomolecules, including cell-wall peptidoglycan precursors (Figure 1 A) ( 1 ), cyclodipeptides ( 2 ) and membrane lipids ( 3 ). In ad- dition, Phe-tRNA and Leu-tRNA participate in protein aminoacylation, thereby triggering them for protein degra- dation by the proteasome ( 4 ). In Staphylococcus aureus, three aminoacyl-transferases of the Fem family (FmhB, FemA and FemB) use five aminoacyl-tRNAs to assemble a pentaglycine side chain onto peptidoglycan precursors ( 5– 7 ). FmhB adds the first Gly whereas FemA and FemB each adds two residues. FmhB is an essential enzyme indicating that the complete absence of the peptidoglycan side chain is not compatible with synthesis of an osmoprotective pepti- doglycan layer and bacterial growth ( 7 ). FemA and FemB are dispensable for growth in media of high osmolarity ( 8 ) and their absence is not compatible with expression of me- thicillin resistance mediated by the peptidoglycan transpep- tidase PBP2a ( 9 ). In fact, the ‘Fem’ designation originates from early investigations based on random transposon mu- tagenesis that showed that the femA and femB genes encode factors essential for methicillin resistance ( 10 ). The essential role of the Fem transferases for growth or for methicillin re- sistance indicates that these enzymes are potential targets to develop drugs active on multidrug resistant S. aureus ( 11– 13 ).
shown using the field stimulation and field recording methods that different inputs can compete for PrPs when the protein pool was made limited ( Fonseca et al., 2004 ) by the application of a translation inhibitor. However, it remains to be determined whether the protein pool would indeed be limiting under more physiological conditions (i.e., in the absence of the translation inhibitor). We also wanted to use our single-spine methodology to examine the temporal dynamics of individual spine changes during situations in which multiply stimulated spines might compete for limiting PrPs. For these purposes, we stimulated two spines between 10 and 20 mm apart on the same branch (labeled L1 and L2) 1 min apart with GLU+FSK stimulation, with the intention of increasing the number of stimulated spines until L-LTP expression was inhibited. We found that stimulating only two spines already caused both spines to reach their maximum volume slower as compared to stimulating only one spine ( Fig- ure 5 A compared to Figure 1 B and Figures S1 A, and S1B, quan- tified in Figure 5 B). Analyzing the temporal dynamics of the competing spines, we found that during the first 30 min, there was a large correlated fluctuation during the poststimulation period in the volumes of L1 and L2, such that the growth of one was accompanied by a shrinking of the other (examples shown in Figure 5 C and Figures S4 B–S4D, quantified in Figure 5 D). In contrast, there was no significant correlation during the baseline period and during time points 40 min or longer after stimulation. In addition, there was also no correlation between stimulated spines and unstimulated neighboring spines ( Fig- ure 5 E) indicating that the competition is specific to stimulated spines. These data suggest that the amount of protein that can be produced within a dendritic compartment at a certain time is limited such that two spines stimulated close together in space and time may compete for available proteins and, hence, for the expression of L-LTP. This might occur due to the relatively limited translational machinery and/or mRNA at the dendritic branch (as compared to the soma) ( Schuman et al., 2006 ). Activity-induced mRNA degradation may also contribute to this phenomenon ( Giorgi et al., 2007 ). These results also suggest that spine growth is a bidirectional rather than a unidirectional dynamic process.