Low Rate Of Protein Synthesis Research Articles

Physiology of trans-translation deficiency in Bacillus subtilis - a comparative proteomics study.

trans-Translation is the most effective ribosome rescue system known in bacteria. While it is essential in some bacteria, Bacillus subtilis possesses two additional alternative ribosome rescue mechanisms that require the proteins BrfA or RqcH. To investigate the physiology of trans-translation deficiency in the model organism B.subtilis, we compared the proteomes of B. subtilis168 and a ΔssrA mutant in the mid-log phase using gel-free label-free quantitative proteomics. In chemically defined medium, the growth rate of the ssrA deletion mutant was 20% lower than that of B.subtilis 168. An 35 S-methionine incorporation assay demonstrated that protein synthesis rates were also lower in the ΔssrA strain. Alternative rescue factors were not detected. Among the 34 proteins overrepresented in the mutant strain were eight chemotaxis proteins. Indeed, both on LB agar and minimal medium the ΔssrA strain showed an altered motility and chemotaxis phenotype. Despite the lower growth rate, in the mutant proteome ribosomal proteins were more abundant while proteins related to amino acid biosynthesis were less abundant than in the parental strain. This overrepresentation of ribosomal proteins coupled with a lower protein synthesis rate and down-regulation of precursor supply reflects the slow ribosome recycling in the trans-translation-deficient mutant.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Life in the freezer: protein metabolism in Antarctic fish.

Whole-animal, in vivo protein metabolism rates have been reported in temperate and tropical, but not Antarctic fish. Growth in Antarctic species is generally slower than lower latitude species. Protein metabolism data for Antarctic invertebrates show low rates of protein synthesis and unusually high rates of protein degradation. Additionally, in Antarctic fish, increasing evidence suggests a lower frequency of successful folding of nascent proteins and reduced protein stability. This study reports the first whole-animal protein metabolism data for an Antarctic fish. Groups of Antarctic, Harpagifer antarcticus, and temperate, Lipophrys pholis, fish were acclimatized to a range of overlapping water temperatures and food consumption, whole-animal growth and protein metabolism measured. The rates of protein synthesis and growth in Antarctic, but not temperate fish, were relatively insensitive to temperature and were significantly lower in H. antarcticus at 3°C than in L. pholis. Protein degradation was independent of temperature in H. antarcticus and not significantly different to L. pholis at 3°C, while protein synthesis retention efficiency was significantly higher in L. pholis than H. antarcticus at 3°C. These results suggest Antarctic fish degrade a significantly larger proportion of synthesized protein than temperate fish, with fundamental energetic implications for growth at low temperatures.

Abstract PO-088: Classification based on efficiency of mRNA translation reveals a metabolically-dependent subtype of pancreatic cancer

Abstract Molecular profiling of Pancreatic Ductal Adenocarcinoma (PDA), based on transcriptomic analyses, identifies two main prognostic subtypes (basal-like and classical), but does not allow personalized first-line treatment. To date, tumors have not been profiled based on protein synthesis rates, yet the step of mRNA translation is highly dysregulated in both PDA cancer cells and their microenvironment. We aim at assessing whether quantification of mRNA translation could provide a distinct perspective on PDA and identify novel tumor subtypes. Using a collection of twenty-seven pancreatic Patient-Derived Xenografts (PDX), we performed transcriptome-wide analysis of translated mRNA (translatome). Unsupervised bioinformatics analysis allowed PDA tumors classification according to mRNA translation rate. PDX-derived cancer cells as well as common PDA cell lines were used to functionally characterize newly identified subtype. Independent component analysis revealed a new tumor subtype harboring a low protein synthesis rate, but associated with a robust translation of mRNAs encoding effectors of the integrated stress response (ISR), including the transcription factor ATF4. Functional characterization of the “ISR-activated” human cancer cells revealed a high resistance to drugs, low autophagic capacities, and importantly, metabolic impairments in the serine synthesis and transsulfuration pathways. Therefore, the drug-resistant cancer cell phenotype showing auxotrophy to both serine and cysteine may be amenable to targeted therapy. Overall, our study highlights profiling of mRNA translation in cancer as an underexplored avenue for identification of previously unrecognized subtypes together with potential treatments. Citation Format: Sauyeun Shin, Remy Nicolle, Mehdi Liauzun, Jacobo Solorzano, Alexia Brunel, Christine Jean, Remi Samain, Jerôme Raffenne, Cindy Neuzillet, Carine Joffre, Stephane Rocci, Juan Iovanna, Nelson Dusetti, Ola Larsson, Stephane Pyronnet, Corinne Bousquet, Yvan Martineau. Classification based on efficiency of mRNA translation reveals a metabolically-dependent subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-088.

Arginase II Plays a Central Role in the Sexual Dimorphism of Arginine Metabolism in C57BL/6 Mice

Sex differences in plasma concentration of arginine and arginase activity of different tissues have been reported in mice. In addition, male but not female C57BL/6 mice have a dietary arginine requirement for growth. The goal of this research was to test the hypothesis that arginase II is a key factor in the sexual dimorphism of arginine metabolism. Young adult male and female wild type (WT), and heterozygous and arginase II knockout mice on a C57BL/6 background mice were infused with labeled citrulline, arginine, ornithine, phenylalanine, and tyrosine to determine the rates of appearance and interconversion of these amino acids. Tissue arginase activity was measured in the liver, heart, jejunum, kidney, pancreas, and spleen with an arginine radioisotope. The effect of genotype, sex, and their interaction was tested. Female mice produced ∼36% more citrulline than their male littermates, which translated into a greater arginine endogenous synthesis, flux, and plasma concentration (42, 6, and 27%, respectively; P< 0.001). Female mice also had a greater phenylalanine flux (10%) indicating a greater rate of whole protein breakdown; however, they had a lower protein synthesis rate than males (18%; P< 0.001). The ablation of arginase II reduced the production of citrulline and the de novo synthesis of arginine in females and increased the rate of appearance of arginine and plasma arginine concentration in male mice (16 and 22%, respectively; P< 0.001). No effect of arginase II deletion, however, was observed for whole-body protein kinetics. Arginase II activity was present in the pancreas, kidney, jejunum, and spleen; WT females had a ∼2-fold greater renal arginase activity than their WT counterparts. A clear sexual dimorphism exists in the endogenous synthesis of arginine and its disposal. Female mice have a greater arginine availability than their male littermates. The ablation of arginase II increases arginine availability in male mice.

Proteostasis and Myeloid Stem Cell Production

Hematopoietic stem cells (HSCs) regenerate blood cells lost to turnover, injury and disease. Defects in HSC maintenance, such as those that occur during aging, lead to anemia, impaired immunity, and bone marrow failure. Over- or ectopic activation of HSC self-renewal programs leads to hematopoietic neoplasms. Thus, defects in HSC maintenance can lead to diverse malignant and non-malignant hematopoietic disorders. We recently discovered that HSCs have lower rates of protein synthesis than other blood cells. Low protein synthesis is necessary for HSCs, as genetic changes that increase protein synthesis impair HSC function. Importantly, this does not simply reflect HSC quiescence, as dividing HSCs also have lower rates of protein synthesis as compared to dividing restricted progenitors. However, why stem cells depend on low protein synthesis and how increases in protein synthesis impair stem cell function remain largely unknown. Translation is a key cog in both the gene expression and protein homeostasis (proteostasis) networks, and thus influences both the content and the quality of the proteome. We have now determined that low protein synthesis within HSCs is associated with elevated proteome quality in vivo. HSCs contain less ubiquitylated and unfolded proteins as compared to restricted myeloid progenitors, and modest increases in protein synthesis cause an accumulation of misfolded/unfolded proteins within HSCs. Thus, HSCs depend upon low protein synthesis to maintain proteome quality. To test how translational control of proteome quality affects stem cell function, we examined Aarssti/sti mice that harbor a mutation in the alanyl-tRNA synthetase, which causes a tRNA editing defect that increases amino acid misincorporation errors during translation. Aarssti/sti mice exhibit reduced HSC numbers and significantly diminished serial reconstituting activity in vivo, but do not exhibit defects within restricted progenitors. Surprisingly, a modest accumulation of misfolded/unfolded proteins does not induce significant activation of the unfolded protein response within HSCs, but instead overwhelms the capacity of the proteasome, which promotes the stabilization and increased abundance of c-Myc. Conditional deletion of a single copy of Myc is sufficient to significantly rescue serial reconstitution defects in Aarssti/sti HSCs. HSCs are thus dependent on low protein synthesis to maintain proteome quality and homeostasis to preserve their self-renewal activity in vivo. Disclosures No relevant conflicts of interest to declare.

Functionally significant metabolic differences between B and T lymphocyte lineages.

Activation of B and T lymphocytes leads to major remodelling of the metabolic landscape of the cells enabling their post-activation functions. However, naive B and T lymphocytes also show metabolic differences, and the genesis, nature and functional significance of these differences are not yet well understood. Here we show that resting B-cells appeared to have lower energy demands than resting T-cells as they consumed lower levels of glucose and fatty acids and produced less ATP. Resting B-cells are more dependent on OXPHOS, while T-cells show more dependence on aerobic glycolysis. However, despite an apparently higher energy demand, T lineage cells showed lower rates of protein synthesis than equivalent B lineage stages. These metabolic differences between the two lineages were established early during lineage differentiation, and were functionally significant. Higher levels of protein synthesis in B-cells were associated with increased synthesis of MHC class II molecules and other proteins associated with antigen internalization, transport and presentation. The combination of higher energy demand and lower protein synthesis in T-cells was consistent with their higher ATP-dependent motility. Our data provide an integrated perspective of the metabolic differences and their functional implications between the B and T lymphocyte lineages.

Dietary Methionine Restriction Upregulates Endogenous H2 S via miR-328-3p: A Potential Mechanism to Improve Liver Protein Metabolism Efficiency in a Mouse Model of High-fat-diet-induced Obesity.

Dietary methionine restriction (MR) promotes multifaceted health benefits. Moreover, lower rate of protein synthesis by dietary MR is associated with life span extension. The goal of this work is to explore how dietary MR would affect protein metabolism in a mouse model of high-fat-diet-induced obesity (DIO). DIO mice (male C57BL/6) are subjected to dietary MR for 22weeks. High-throughput sequencing technology, qRT-PCR analysis, and the dual luciferase reporter assay are performed to verify that MiR-328-3p directly targets cystathionine γ-lyase (CSE) to modulate endogenous H2 S production. Moreover, indicators of endogenous H2 S, fractional synthesis rate (FSR), fractional growth rate (FGR), fractional degradation rate (FDR), and protein retention efficiency (PRE) are analyzed. MR results in an increase in endogenous H2 S to relieve oxidative stress and ER stress to improve protein homeostasis and metabolic efficiency in DIO mice. Results show that dietary MR increases endogenous H2 S production via miR-328-3p. Furthermore, these results suggest the potential involvement of endogenous H2 S on the efficiency of protein metabolism in dietary MR.

Regulation of unfolded protein response in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) play a central role in hematopoietic regeneration, which has been demonstrated by thorough studies. In contrast, the cell cycle status and metabolic condition of HSCs define these cells as dormant. Recent studies have also revealed that protein metabolism is quite unique, as dormant HSCs have a lower protein synthesis rate and folding capacity. Under proliferative conditions, upon hematopoietic stress, HSCs need to deal with higher requirements of protein production to achieve fast and effective blood replenishment. In such cases, increased protein synthesis could exceed the capacity of precise protein quality control, leading to the accumulation of unfolded and misfolded proteins. In turn, this triggers endoplasmic reticulum (ER) stress as a part of the unfolded protein response (UPR). Since ER stress is a multi-layered, bidirectional cellular response that contains both positive (survival) and negative (death) reactions, proper management of UPR and ER stress signals is crucial for HSCs and also for maintaining the healthy hematopoietic system. In this review, we introduce the latest findings in this emerging field within hematopoiesis and HSC regulation.

Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised proteinday-1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate.

Energy restriction upregulates circulating myomiR expression in vivo and in vitro

Energy restriction (ER)‐induced weightloss attenuates the rate of protein synthesis, which over time can result inreductions in skeletal muscle mass. Recently, changes in the expression of circulating muscle‐specific microRNA (c‐myomiR)have been suggested to reflect rates of protein synthesis. However, whether an increased expression ofc‐myomiR are associated with lower rates of protein synthesis remains undefined. The present investigation sought to determine whether ER influences c‐myomiR expression, and if changes in c‐myomiR are associated with rates of protein synthesis both in vivo (humans) and in vitro (cultured myocytes). Sixteen older (64 ± 2 yrs) overweight (28.5 ±1.2 kg·m−2) males enrolled in this 35‐day controlled feedingtrial. A 7‐day weight maintenance (WM)period was followed by 28 days of 30% ER; whole‐body protein turnover (15N‐glycine)and c‐myomiR (miR‐1–3p, miR‐133a‐3p, miR‐133b, miR‐206) expression was assessed by RT‐qPCR at the conclusions of WM and ER. Participants lost 4.4 ± 0.3 kg body mass during ER (P &lt; 0.05). Overall, ER upregulated c‐myomiR expression compared to WM (P &lt; 0.05). Expression ofc‐myomiR was inversely associated (r= −0.700, P &lt; 0.05) with whole‐body protein synthesis after ER. Similarly, assessing the expression of c‐myomiR obtained from media that C2C12myotubes were cultured; ER increased c‐myomiR expression by 2.9 ± 0.1 fold and lowered the protein synthesis rate by 2.1 ±0.2 fold compared to control. Corroboration of in vitro and in vivo findings indicates that increased expressions of c‐myomiR maybe noninvasive markers reflective of ER‐induced reductions in the rate protein synthesis.Support or Funding InformationThis material is based on the work supported by the U.S. Department of Agriculture(USDA), under agreement No. 58‐1950‐4‐003 and the US Army Medical Research and Material Command. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the USDA. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The study was also supported by the Boston Claude D. Pepper Center Older American Independence Centers (OAIC;1P30AG031679). L.M.M is supported by T32NIDDK training grant # 5T32DK062032‐23. D.A.R. is supported by NIA K01 award # KAG047247A‐A1.

Abstract IA12: Low rates of protein synthesis in hematopoietic stem cells are determined partly by relatively high levels of hypophosphorylated eIF4E-BP

Abstract Hematopoietic stem cells (HSCs) synthesize less protein per hour, on average, than other hematopoietic progenitors. Limited protein synthesis appears to be a widely shared feature of adult stem cells. The low rate of protein synthesis is necessary for HSC maintenance as genetic changes that modestly increase or decrease protein synthesis each impair HSC function. However, the mechanisms that suppress protein synthesis in adult stem cells remain unexplored. We assessed a number of potential mechanisms and found that the reduced rate of protein synthesis in HSCs as compared to other hematopoietic progenitors is not explained by differences in proteasome activity, cell division rate, or mRNA content. However, adult bone marrow HSCs had increased amounts of hypophosphorylated eukaryotic initiation factor 4E binding protein1 (eIF4E-BP1) and eIF4E-BP2 as compared to other hematopoietic progenitors. Since unphosphorylated eIF4E-BP negatively regulates protein synthesis, this raised the possibility that eIF4E-BPs hypophosphorylation suppresses protein synthesis in HSCs. Consistent with this, genetic inactivation of eIF4E-BP1 and eIF4E-BP2 increased protein synthesis in HSCs and impaired their reconstituting activity. These findings provide insight into the mechanisms that promote adult tissue regenerative capacity by attenuating protein synthesis in stem cells. Citation Format: Robert A. J. Signer, Le Qi, Sean J. Morrison. Low rates of protein synthesis in hematopoietic stem cells are determined partly by relatively high levels of hypophosphorylated eIF4E-BP. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA12.

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.

The low rate of protein synthesis in hematopoietic stem cells is determined partly by hypophosphorylation of 4E-BPS

The low rate of protein synthesis in hematopoietic stem cells is determined partly by hypophosphorylation of 4E-BPS

Phosphorylation of eIF2α on Threonine 169 is not required for Trypanosoma brucei cell cycle arrest during differentiation

The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating ‘slender' bloodstream forms differentiate to non-dividing ‘stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.

Protein Requirements and Recommendations for Older People: A Review.

Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise). Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV) that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

Epinephrine depletion exacerbates the fasting-induced protein breakdown in fast-twitch skeletal muscles

The physiological role of epinephrine in the regulation of skeletal muscle protein metabolism under fasting is unknown. We examined the effects of plasma epinephrine depletion, induced by adrenodemedullation (ADMX), on muscle protein metabolism in fed and 2-day-fasted rats. In fed rats, ADMX for 10 days reduced muscle mass, the cross-sectional area of extensor digitorum longus (EDL) muscle fibers, and the phosphorylation levels of Akt. In addition, ADMX led to a compensatory increase in muscle sympathetic activity, as estimated by the rate of norepinephrine turnover; this increase was accompanied by high rates of muscle protein synthesis. In fasted rats, ADMX exacerbated fasting-induced proteolysis in EDL but did not affect the low rates of protein synthesis. Accordingly, ADMX activated lysosomal proteolysis and further increased the activity of the ubiquitin (Ub)-proteasome system (UPS). Moreover, expression of the atrophy-related Ub ligases atrogin-1 and MuRF1 and the autophagy-related genes LC3b and GABARAPl1 were upregulated in EDL muscles from ADMX-fasted rats compared with sham-fasted rats, and ADMX reduced cAMP levels and increased fasting-induced Akt dephosphorylation. Unlike that observed for EDL muscles, soleus muscle proteolysis and Akt phosphorylation levels were not affected by ADMX. In isolated EDL, epinephrine reduced the basal UPS activity and suppressed overall proteolysis and atrogin-1 and MuRF1 induction following fasting. These data suggest that epinephrine released from the adrenal medulla inhibits fasting-induced protein breakdown in fast-twitch skeletal muscles, and these antiproteolytic effects on the UPS and lysosomal system are apparently mediated through a cAMP-Akt-dependent pathway, which suppresses ubiquitination and autophagy.

English

&nbsp; Previous studies have shown that hydrolyzed proteins exhibit antioxidant properties and may confer physical and physiological advantages when consumed by the exercising rat. The purpose of this study was to compare the effects of feeding either intact (I) and partly hydrolyzed (H) milk whey proteins on gastrocnemial DNA contents and protein metabolism in exercising Wistar rats. Protein synthesis and degradation, protein and DNA contents, and concentration of the serum insulin-like growth factor-1 (IGF1) were determined in six experimental groups according to the type of protein consumed [casein (C), whey protein isolate (I), hydrolyzed whey protein (H) and level of physical activity (sedentary (S) and trained (T)]. H produced significantly lower rates of protein synthesis and degradation and DNA contents in the gastrocnemius, while no differences were observed in the total muscle protein content and serum levels of IGF1. These results indicate that consumption of prehydrolyzed whey protein alters muscle metabolism resulting in less DNA, but maintains the muscle protein levels constant and sustain or improves physical performance, compared to the unhydrolyzed protein. &nbsp; Key words:&nbsp;Dietary protein, hydrolyzed whey protein, peptides in muscle metabolism, phenylalanine, tyrosine, physical activity.

Proline Supplementation to Parenteral Nutrition Results in Greater Rates of Protein Synthesis in the Muscle, Skin, and Small Intestine in Neonatal Yucatan Miniature Piglets1–3

Proline and arginine are each indispensable during parenteral feeding due to limited interconversion by an atrophied gut. Commercial amino acid parenteral products designed for neonates contain proline concentrations that differ by almost 4-fold. To assess the adequacy of the lowest concentration of proline provided in commercial total parenteral nutrition (TPN) products, we compared rates of tissue-specific protein synthesis and nitrogen balance in neonatal piglets provided TPN at 2 different proline concentrations. Yucatan miniature piglets (9–11 d old, n = 12) were randomized to complete isonitrogenous TPN diets with low proline (LP; L-proline as 3% of amino acids) or proline supplemented (PS; 9%). After 7 d of receiving TPN, rates of protein synthesis in liver, gastrocnemius muscle, jejunal mucosa, and skin were determined by the flooding dose technique and tissue free amino acids were measured. Nitrogen balance was assessed during the last 3 d. The LP TPN resulted in lower free proline concentrations in plasma, muscle, and skin (P < 0.05) and lower rates of protein synthesis in the jejunum (by 25%; P = 0.02), muscle (by 45%; P = 0.015), and skin (by 60%; P = 0.01); there was no difference in liver. Nitrogen retention was 20% lower in the LP group (P = 0.01). In conclusion, muscle and skin protein synthesis was profoundly sensitive to parenteral proline supply and the reduced protein synthesis in the intestine could affect intestinal integrity. Low-proline TPN solutions that are currently in wide use in neonatal care may result in impaired tissue growth.

Measurement of the rate of protein turnover and synthesis in the marsupial Honey possum (Tarsipes rostratus)

Rates of protein turnover and synthesis were measured in wild-caught Honey possums (Tarsipes rostratus) in the southwest of Western Australia and compared between males and females with and without pouch young. Possums were injected with 50 microg of (15)N-glycine and ammonia collected within 24 h was used as the nitrogen end-product in a single-injection protocol. The overall mean rate of protein synthesis measured was 7.7+/-0.5 g kg(-0.75) day(-1), which falls within the range of values reported for other marsupial species. Whole body rates of nitrogen flux and protein synthesis did not vary significantly between males and females with and without young, but females with pouch young showed significantly lower rates of protein synthesis when expressed in relation to metabolic body size. This difference was no longer apparent, however, if the mass of the females was corrected for the estimated mass of the young in the pouch averaging 9.3+/-1.6 g kg(-0.75) day(-1) and suggesting that the young should not be considered as part of the metabolic body pool. Whole body rates of protein degradation were significantly reduced in females carrying pouch young, suggesting that protein may be being diverted from the pool to milk production. Calculations indicate that the daily fraction of the female's nitrogen synthesis rate that needs to be diverted to pouch young to sustain their growth is less than 5%, and may not be detectable with the current methodology.