Essential Amino Acid-Enriched Diet Alleviates Dexamethasone-Induced Loss of Muscle Mass and Function through Stimulation of Myofibrillar Protein Synthesis and Improves Glucose Metabolism in Mice

Dexamethasone (DEX) induces dysregulation of protein turnover, leading to muscle atrophy and impairment of glucose metabolism. Positive protein balance, i.e., rate of protein synthesis exceeding rate of protein degradation, can be induced by dietary essential amino acids (EAAs). In this study, we investigated the roles of an EAA-enriched diet in the regulation of muscle proteostasis and its impact on glucose metabolism in the DEX-induced muscle atrophy model. Mice were fed normal chow or EAA-enriched chow and were given daily injections of DEX over 10 days. We determined muscle mass and functions using treadmill running and ladder climbing exercises, protein kinetics using the D2O labeling method, molecular signaling using immunoblot analysis, and glucose metabolism using a U-13C6 glucose tracer during oral glucose tolerance test (OGTT). The EAA-enriched diet increased muscle mass, strength, and myofibrillar protein synthesis rate, concurrent with improved glucose metabolism (i.e., reduced plasma insulin concentrations and increased insulin sensitivity) during the OGTT. The U-13C6 glucose tracing revealed that the EAA-enriched diet increased glucose uptake and subsequent glycolytic flux. In sum, our results demonstrate a vital role for the EAA-enriched diet in alleviating the DEX-induced muscle atrophy through stimulation of myofibrillar proteins synthesis, which was associated with improved glucose metabolism.

Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila

Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. Our recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. Here, we use this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, our results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. Our models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, our modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.

DnaK response to expression of protein mutants is dependent on translation rate and stability

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the lambda-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

AGING & MUSCULAR FUNCTION: A SELECTED REVIEW OF LITERATURE WITH EMPHASIS ON CARDIORESPIRATORY ENDURANCE AND FUNCTIONAL PERFORMANCE RESPONSE TO RESISTANCE EXERCISE

This narrative review evaluates strength or resistance training on cardiorespiratory endurance, blood pressure, contractile function, contractile protein synthesis rate, bone turnover, gait and balance, and neuromuscular adaptations in elderly populations. Seventy-eight studies spanning from 1999 through 2020 were reviewed. Database sources including PubMed, Science Direct, Web of Knowledge and Google Scholar were searched in accordance with the purpose of the study. A majority of the studies reported that resistance training reduces blood pressure and increases contractile functions, contractile protein synthesis rate, bone turnover, gait and balance, cardiorespiratory endurance, and neuromuscular adaptations in the elderly. Furthermore, combined training (CT), also known as concurrent training (strength plus endurance training) may also be as effective as traditional endurance training or traditional strength/resistance training alone for improving cardiorespiratory endurance and functional performance. According to the evaluation of studies included in this review, we concluded that training modalities that involve low-load, high velocity strength training combined with endurance training might be the best training strategy in improving cardiovascular fitness, functional capacity and musculoskeletal health in the elderly populations. Elderly people should be encouraged to participate in a concurrent training or a combination of strength and endurance training to delay, or even reverse the negative effects of aging. <p> </p><p><strong> Article visualizations:</strong></p><p><img src="/-counters-/edu_01/0875/a.php" alt="Hit counter" /></p>

Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

Background Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results This study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. Conclusions We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

Impaired Muscle Mitochondrial Function in Familial Partial Lipodystrophy

Background Familial Partial Lipodystrophy (FPL), Dunnigan variety is characterized by skeletal muscle hypertrophy and insulin resistance besides fat loss from the extremities. The cause for the muscle hypertrophy, and its functional consequences is not known. Objective To compare muscle strength and endurance, besides muscle protein synthesis rate between subjects with FPL and matched controls (n = 6 in each group). In addition, we studied skeletal muscle mitochondrial function and gene expression pattern to help understand the mechanisms for the observed differences. Methods Body composition by DEXA, insulin sensitivity by minimal modelling, assessment of peak muscle strength and fatigue, skeletal muscle biopsy and calculation of muscle protein synthesis rate, mitochondrial respirometry, skeletal muscle transcriptome, proteome and gene set enrichment analysis. Results Despite increased muscularity, FPL subjects did not demonstrate increased muscle strength but had earlier fatigue on chest press exercise. Decreased mitochondrial state 3 respiration in the presence of fatty acid substrate was noted, concurrent to elevated muscle lactate and decreased long-chain acylcarnitine. Based on gene transcriptome, there was significant down regulation of many critical metabolic pathways involved in mitochondrial biogenesis and function. Moreover, the overall pattern of gene expression was indicative of accelerated aging in FPL subjects. A lower muscle protein synthesis and down regulation of gene transcripts involved in muscle protein catabolism was observed. Conclusion Increased muscularity in FPL is not due to increased muscle protein synthesis and is likely due to reduced muscle protein degradation. Impaired mitochondrial function and altered gene expression likely explain the metabolic abnormalities and skeletal muscle dysfunction in FPL subjects.

DnaK response to expression of protein mutants is dependent on translation rate and stability

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the λ-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

The Gelation of CAG Repeat Expansion RNAs Suppresses Global Protein Translation

RNA molecules with the expanded CAG repeat (eCAGr) may undergo liquid-to-gel phase transitions rapidly, but the nuclear eCAGr RNA foci display liquid-like properties, different from their gel-like behaviour in vitro (ref.1). The functional impact of this RNA gelation is also completely unknown (ref.2). Here we demonstrate that eCAGr RNA may form gel-like condensates (foci) in the cytoplasm that were rapidly degraded by lysosomes in a LAMP2-dependent manner. These RNA foci may lead to a drastic reduction of the global protein synthesis rate in cells and in vitro, possibly by sequestering the key protein translation elongation factor eEF2, which formed puncta colocalizing or surrounding the cytoplasmic eCAGr RNA condensates. Disrupting the eCAGr RNA gelation partially restored the global protein translation rate whereas the induction of enhanced gelation by an optogenetic system exacerbated this phenotype. Finally, eEF2 puncta were significantly enhanced in brain slices from a mouse model and patients of Huntington’s disease, which is a CAG expansion disorder expressing eCAGr RNAs. Our study demonstrates the RNA gelation inside the cells and reveals its functional impact, providing new angles for understanding pathological mechanisms of repeat expansion diseases and global protein translation regulation.

The Effects of Age and Adiposity on Casein Digestibility and Tissue Protein Synthesis in Rats

Objectives Age and adiposity can impact the digestibility of dietary proteins and the metabolic response to their ingestion. The objective was to evaluate the effects of age and adiposity on casein digestibility and protein synthesis in tissues and organs. Methods Wistar rats of 1 month (n = 15) and 10 months (n = 15) at their arrival were fed ad libitum with a standard diet or High Fat diet to obtain rats of normal and high adiposity levels. Four groups were constituted (n = 7/8): 2 months/normal adiposity, 2 months/high adiposity, 11 months/normal adiposity and 11 months/high adiposity. At the end of the dietary intervention, they were fed the standard diet for 1 week before the test meal. The rats consumed a 4g meal containing 15N-labeled casein (Prodiet® 85B). Six hours after meal ingestion, the rats were euthanized. Intravenous injection of a massive dose of 13C-valine prior to euthanasia was used to determine protein synthesis rate in liver, kidneys, skin and muscle. Body composition was evaluated and digestive contents were collected to measure casein digestibility. Results No weight difference between rats of the same age was observed. However, a significant difference in adiposity was noted, with a surge in body fat of 3% in young rats and 7% in older rats. Digestibility increased with a higher adiposity level (P = 0.04). In young rats, it was 94.1 ± 1.1% in lean rats and 95.2 ± 1.7% in fat rats. In older rats, it was 94.5 ± 2.2% and 95.8 ± 0.7%, in lean and fat rats respectively. Significant effects of age (P &lt; 0.01) and adiposity (P &lt; 0.01) were observed in the muscle fractional synthesis rate (FSR), with age decreasing it and adiposity increasing it. In young rats, FSR was 10.1 ± 2.1%/day and 12.0 ± 3.0%/day in lean and fat rats, respectively, these values being 6.2 ± 1.5%/day and 10.6 ± 2.0%/day in older rats. In the skin, younger rats exhibited a higher FSR (P &lt; 0.01) as it was 11.1 ± 2.6%/day and 12.6 ± 3.7%/day in lean and fat rats respectively, and 8.3 ± 2.3%/day and 8.2 ± 2.7%/day in older rats. No differences were found for the liver and kidneys. Conclusions Protein synthesis in muscle decreased with age while adiposity increased it. This is consistent with an improvement in ribosomal activity at an intermediate state of obesity. The surge in casein digestibility with higher adiposity, although moderate, could have contributed to the improvement in muscle anabolism response. Funding Sources Ingredia.