Plant Protein Can Be as Efficient as Milk Protein to Maintain Fat Free Mass in Old Rats, Even When Fat and Sugar Intakes Are High.

BACKGROUND: Alternative, sustainable, and adequate sources of protein must be found to meet global demand. OBJECTIVES: Our aim was to assess the effect of a plant protein blend with a good balance of indispensable amino acids and high contents of leucine, arginine, and cysteine on the maintenance of muscle protein mass and function during aging in comparison to milk proteins and to determine if this effect varied according to the quality of the background diet. METHODS: Old male Wistar rats (n = 96, 18 mo old) were randomly allocated for 4 mo to 1 of 4 diets, differing according to protein source (milk or plant protein blend) and energy content (standard, 3.6 kcal/g, with starch, or high, 4.9 kcal/g, with saturated fat and sucrose). We measured: every 2 mo, body composition and plasma biochemistry; before and after 4 mo, muscle functionality; after 4 mo, in vivo muscle protein synthesis (flooding dose of L-[1-13C]-valine) and muscle, liver, and heart weights. Two-factor ANOVA and repeated measures 2-factor ANOVA were conducted. RESULTS: There was no difference between protein type on the maintenance during aging of lean body mass, muscle mass, and muscle functionality. The high-energy diet significantly increased body fat (+47%) and heart weight (+8%) compared to the standard energy diet but had no effect on fasting plasma glucose and insulin. Muscle protein synthesis was significantly stimulated by feeding to the same extent in all groups (+13%). CONCLUSIONS: Since high-energy diets had little impact on insulin sensitivity and related metabolism, we could not test the hypothesis that in situations of higher insulin resistance, our plant protein blend may be better than milk protein. However, this rat study offers significant proof of concept from the nutritional standpoint that appropriately blended plant proteins can have high nutritional value even in demanding situations such as aging protein metabolism.

Anti-inflammatory Streptococcus thermophilus CNRZ160 limits sarcopenia induced by low-grade inflammation in older adult rats.

Background and aims: Aging is characterized, at the systemic level, by the development of low-grade inflammation, which has been identified as determining sarcopenia by blunting postprandial muscle anabolism. The causes of this "inflammageing" is still not clearly defined. An increased intestinal permeability, a microbiota dysbiosis and subsequent generation of intestinal then generalized inflammation have been hypothesized. The objective of this study was to test in vivo during aging if (1) a chronic low-grade intestinal inflammation can lead to anabolic resistance and muscle loss and (2) if a bacterial strain presenting anti-inflammatory properties could prevent these adverse effects. Methods: Young adult (6 m) and elderly rats (18 m) received Dextran Sodium Sulfate (DSS) for 28 days to generate low-grade intestinal inflammation, and received (PB1 or PB2 groups) or not (DSS group) one of the two S. Thermophilus strains (5 × 109 CFU/day) previously shown to present an anti-inflammatory potential in vitro. They were compared to pair fed control (PF). Muscle and colon weights and protein synthesis (using 13C Valine) were measured at slaughter. Muscle proteolysis, gut permeability and inflammatory markers were assessed only in old animals by RT-PCR or proteins quantifications (ELISA). Results: In both adult and old rats, DSS reduced absolute protein synthesis (ASR) in gastrocnemius muscle [-12.4% (PB1) and -9.5% (PB2) vs. PF, P < 0.05] and increased ASR in colon (+86% and +30.5%, respectively vs. PF, P < 0.05). PB1 (CNRZ160 strain) but not PB2 resulted in a higher muscle ASR as compared to DSS in adults (+18%, P < 0.05), a trend also observed for PB1 in old animals (+12%, P = 0.10). This was associated with a blunted increase in colon ASR. In old rats, PB1 also significantly decreased expression of markers of autophagy and ubiquitin-proteasome pathways vs. DSS groups and improved gut permeability (assessed by Occludin, Zonula Occludens 1 and Claudin 1 expression, P < 0.05) and alleviated systemic inflammation (A2M: -48% vs. DSS, P < 0.05). Conclusion: The loss of muscle anabolism associated with low-grade intestinal inflammation can be prevented by supplementation with anti-inflammatory CNRZ160 strain. We propose that the moderated gut inflammation by CNRZ160 may result in curtailed amino acids (AA) utilization by the gut, and subsequent restored AA systemic availability to support muscle protein accretion. Therefore, CNRZ160 could be considered as an efficient probiotic to modulate muscle mass loss and limit sarcopenia during aging.

Lacticaseibacillus casei CNCM I-5663 supplementation maintained muscle mass in a model of frail rodents.

The aim of this study was to identify a probiotic-based strategy for maintaining muscle anabolism in the elderly. In previous research, we found that individuals experiencing short bowel syndrome (SBS) after an intestinal resection displayed beneficial metabolic adjustments that were mediated by their gut microbes. Thus, these bacteria could potentially be used to elicit similar positive effects in elderly people, who often have low food intake and thus develop sarcopenia. Gut bacterial strains from an SBS patient were evaluated for their ability to (1) maintain Caenorhabditis elegans survival and muscle structure and (2) promote protein anabolism in a model of frail rodents (18-month-old rats on a food-restricted diet: 75% of ad libitum consumption). We screened a first set of bacteria in C. elegans and selected two Lacticaseibacillus casei strains (62 and 63) for further testing in the rat model. We had four experimental groups: control rats on an ad libitum diet (AL); non-supplemented rats on the food-restricted diet (R); and two sets of food-restricted rats that received a daily supplement of one of the strains (∼109 CFU; R+62 and R+63). We measured lean mass, protein metabolism, insulin resistance, cecal short-chain fatty acids (SCFAs), and SCFA receptor expression in the gut. Food restriction led to decreased muscle mass [-10% vs. AL (p < 0.05)]. Supplementation with strain 63 tempered this effect [-2% vs. AL (p > 0.1)]. The mechanism appeared to be the stimulation of the insulin-sensitive p-S6/S6 and p-eIF2α/eIF2α ratios, which were similar in the R+63 and AL groups (p > 0.1) but lower in the R group (p < 0.05). We hypothesize that greater SCFA receptor sensitivity in the R+63 group promoted gut-muscle cross talk [GPR41: +40% and GPR43: +47% vs. R (p < 0.05)]. Hence, strain 63 could be used in association with other nutritional strategies and exercise regimes to limit sarcopenia in frail elderly people.

Corrigendum: Lacticaseibacillus casei CNCM I-5663 supplementation maintained muscle mass in a model of frail rodents.

[This corrects the article DOI: 10.3389/fnut.2022.928798.].

Post Meal Energy Boluses Do Not Increase the Duration of Muscle Protein Synthesis Stimulation in Two Anabolic Resistant Situations.

BACKGROUND: 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 or catabolic states. It has been hypothesized 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. Interestingly, it has been shown recently that the duration of the postprandial stimulation of muscle protein synthesis in healthy conditions can be prolonged by the supplementary ingestion of a desynchronized carbohydrate load after food intake. We verified this hypothesis in the present study in two different cases of muscle wasting associated with anabolic resistance, i.e., glucocorticoid treatment and aging. METHODS: Multi-catheterized minipigs were treated or not with glucocorticoids for 8 days. Muscle protein synthesis was measured sequentially over time after the infusion of a 13C phenylalanine tracer using the arterio-venous method before and after whey protein meal ingestion. The energy bolus was given 150 min after the meal. For the aging study, aged rats were fed the whey meal and muscle protein synthesis was measured sequentially over time with the flooding dose method using 13C Valine. The energy bolus was given 210 min after the meal. RESULTS: Glucocorticoid treatment resulted in a decrease in the duration of the stimulation of muscle protein synthesis. The energy bolus given after food intake was unable to prolong this stimulation despite a simultaneous increase of insulin and glucose following its absorption. In old rats, a similar observation was made with no effect of the energy bolus on the duration of the muscle anabolic response following whey protein meal intake. CONCLUSIONS: Despite very promising observations in healthy situations, the strategy aimed at increasing muscle protein synthesis stimulation by giving an energy bolus during the postprandial period remained inefficient in our two anabolic resistance models.

A meal with mixed soy/whey proteins is as efficient as a whey meal in counteracting the age-related muscle anabolic resistance only if the protein content and leucine levels are increased.

With aging, skeletal muscle becomes resistant to the anabolic effect of dietary proteins and sarcopenia develops. Animal proteins, which are rich in leucine, are recommended for the elderly, but it is not known whether their replacement by plant proteins would maintain the health and physical independence of this population. Aged rats were fed with animal proteins (casein and whey proteins) with different leucine contents and compared to rats fed with diets in which whey was substituted with soy proteins and by increasing the total protein content or not. Our results clearly showed that the meal with mixed soy/whey proteins allowed the anabolic response of skeletal muscle during aging only if the protein content was increased by 25%. Indeed, if the protein content of the soy/whey diet was decreased to a similar protein content such as a whey diet, i.e. 13%, the anabolic effect decreased. The same observation was recorded if the whey proteins were totally substituted with soy proteins.

At same leucine intake, a whey/plant protein blend is not as effective as whey to initiate a transient post prandial muscle anabolic response during a catabolic state in mini pigs.

BACKGROUND: Muscle atrophy has been explained by an anabolic resistance following food intake and an increase of dietary protein intake is recommended. To be optimal, a dietary protein has to be effective not only to initiate but also to prolong a muscle anabolic response in a catabolic state. To our knowledge, whether or not a dairy or a dairy/plant protein blend fulfills these criterions is unknown in a muscle wasting situation. OBJECTIVE: Our aim was, in a control and a catabolic state, to measure continuously muscle anabolism in term of intensity and duration in response to a meal containing casein (CAS), whey (WHEY) or a whey/ plant protein blend (BLEND) and to evaluate the best protein source to elicit the best post prandial anabolism according to the physio-pathological state. METHODS: Adult male Yucatan mini pigs were infused with U-13C-Phenylalanine and fed either CAS, WHEY or BLEND. A catabolic state was induced by a glucocorticoid treatment for 8 days (DEX). Muscle protein synthesis, proteolysis and balance were measured with the hind limb arterio-venous differences technique. Repeated time variance analysis were used to assess significant differences. RESULTS: In a catabolic situation, whey proteins were able to initiate muscle anabolism which remained transient in contrast to the stimulated muscle protein accretion with WHEY, CAS or BLEND in healthy conditions. Despite the same leucine intake compared to WHEY, BLEND did not restore a positive protein balance in DEX animals. CONCLUSIONS: Even with WHEY, the duration of the anabolic response was not optimal and has to be improved in a catabolic state. The use of BLEND remained of lower efficiency even at same leucine intake than whey.

UBE2D2 is not involved in MuRF1-dependent muscle wasting during hindlimb suspension.

The Ubiquitin Proteasome System (UPS) is mainly responsible for the increased protein breakdown observed in muscle wasting. The E3 ligase MuRF1 is so far the only enzyme known to direct the main contractile proteins for degradation (i.e. troponin I, myosin heavy chains and actin). However, MuRF1 does not possess any catalytic activity and thus depends on the presence of a dedicated E2 for catalyzing the covalent binding of polyubiquitin (polyUb) chains on the substrates. The E2 enzymes belonging to the UBE2D family are commonly used for in vitro ubiquitination assays but no experimental data suggesting their physiological role as bona fide MuRF1-interacting E2 enzymes are available. In this work, we first found that the mRNA levels of critical E3 enzymes implicated in the atrophying program (MuRF1, MAFbx, Nedd4 and to a lesser extent Mdm2) are tightly and rapidly controlled during the atrophy (up regulation) and recovery (down regulation) phases in the soleus muscle from hindlimb suspended rats. By contrast, E3 ligases (Ozz, ASB2ß and E4b) implicated in other processes (muscle development or regeneration) poorly responded to atrophy and recovery. UBE2B, an E2 enzyme systematically up regulated in various catabolic situations, was controlled at the mRNA levels like the E3s implicated in the atrophying process. By contrast, UBE2D2 was progressively repressed during atrophy and recovery, which makes it a poor candidate for a role during muscle atrophy. In addition, UBE2D2 did not exhibit any affinity with MuRF1 using either yeast two-hybrid or Surface Plasmon Resonance (SPR) approaches. Finally, UBE2D2 was unable to promote the degradation of the MuRF1 substrate α-actin in HEK293T cells, suggesting that no functional interaction exists between these enzymes within a cellular context. Altogether, our data strongly suggest that UBE2D2 is not the cognate ubiquitinating enzyme for MuRF1 and that peculiar properties of UBE2D enzymes may have biased in vitro ubiquitination assays.

UBE2B is implicated in myofibrillar protein loss in catabolic C2C12 myotubes.

BACKGROUND: Skeletal muscle protein loss is an adaptive response to various patho-physiological situations, and the ubiquitin proteasome system (UPS) is responsible for the degradation of the bulk of muscle proteins. The role of E2 ubiquitin-conjugating enzymes is still poorly understood in skeletal muscle. METHODS: We screened for E2s expression levels in C2C12 myotubes submitted to the catabolic glucocorticoid dexamethasone (Dex). RESULTS: One micromolar Dex induced an accumulation of proteasome substrates (polyUb conjugates) and an overexpression of the muscle-specific E3 ligase MuRF1 and of six E2 enzymes, UBE2A, UBE2B, UBE2D1, UBE2D2, UBE2G1, and UBE2J1. However, only MuRF1 and UBE2B were sensitive to mild catabolic conditions (0.16 µM Dex). UBE2B knockdown induced a sharp decrease of total (-18%) and K48 (-28%) Ub conjugates, that is, proteasome substrates, indicating an important role of UBE2B in the overall protein breakdown in catabolic myotubes. CONCLUSIONS: Interestingly, these results indicate an important role of UBE2B on muscle protein homeostasis during catabolic conditions.

Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1.

Muscle atrophy prevails in numerous diseases (cancer cachexia, renal failure, infections, etc.), mainly results from elevated proteolysis, and is accelerated by bed rest. This largely contributes to increased health costs. Devising new strategies to prevent muscle wasting is a major clinical challenge. The ubiquitin proteasome system (UPS) degrades myofibrillar proteins, but the precise mechanisms responsible for actin breakdown are surprisingly poorly characterized. We report that chimeric flag-actin was destabilized and polyubiquitinylated in stably transfected C2C12 myotubes treated with the catabolic agent dexamethasone (1 µM) and that only proteasome inhibitors blocked its breakdown. Actin polyubiquitinylation was also detected in wild-type C2C12 myotubes and human muscle biopsies from control participants and patients with cancer. The muscle-specific E3 ubiquitin ligase MuRF1 is up-regulated in catabolic conditions and polyubiquitinylates components of the thick filament. We also demonstrate that recombinant GST-MuRF1 physically interacted and polyubiquitinylated actin in vitro and that MuRF1 is a critical component for actin breakdown, since MuRF1 siRNA stabilized flag-actin. These data identify unambiguously the abundant contractile protein actin as a target of the UPS in skeletal muscle both in vitro and in vivo, further supporting the need for new strategies blocking specifically the activation of this pathway in muscle wasting conditions.

A new method of purification of proteasome substrates reveals polyubiquitination of 20 S proteasome subunits.

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzyme-mediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified alpha2, alpha6, alpha7, beta2, beta3, beta4, and beta5 20 S proteasome subunits as potential substrates. Western blots using an anti-beta3 proteasome subunit antibody confirmed that high molecular weight forms of beta3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.