Rate Of Protein Synthesis Research Articles

Anabolic Characterization Of Immortalized Murine L6, And Canine Myok9 Skeletal Myotubes

Healthy skeletal muscle is an important site of metabolic regulation. Metabolic dysregulation of skeletal muscle contributes to the development of obesity, the metabolic syndrome, and muscle degeneration. Interest in developing an in vitro canine model has been increasing as canine skeletal muscle is more pathologically similar to human skeletal muscle than rodent. In vitro models are useful to control variables, reduce clinical costs, and assess molecular characteristics of tissue. Therefore, characterization of Myok9 myotube anabolic function and comparison to an established cell line is necessary to understand metabolic function of this novel cell line for its use in the study of skeletal muscle dysfunction and disease. PURPOSE: To describe anabolic signaling and determine fractional synthesis rates of novel Myok9 myotubes when compared to established murine L6 myotubes. METHODS: L6 and Myok9 myoblasts were grown in vitro in standard culture medium until 70% confluent. Differentiation was induced by switching to a low serum medium. Media containing 4% deuterium oxide (stable isotope label) supplemented with the appropriate pharmacological treatment (DMSO, rapamycin, and torin1) was added to myotubes during the final 24-hours of the 7-day differentiation period. Cells and media samples were harvested for fractional synthesis rates, and protein content of key anabolic markers using Western blotting techniques. RESULTS: Myofibrillar protein synthesis rates (MPS) were similar between species, but slightly lower in canine versus rat cells. Both cell lines exhibited significantly lower MPS in the myotubes treated with torin1. The phosphorylated to total protein ratios of 4EBP1 were significantly lower in both L6 and Myok9 myotubes treated with torin1. The phosphorylated to total protein ratios of p70S6K were significantly lower in L6 and Myok9 myotubes treated with rapamycin or torin1. CONCLUSION: Similar anabolic responses to pharmacologic inhibition occurred in both L6 and Myok9 myotubes. Interestingly, MPS rates in Myok9 myotubes were slightly lower compared to L6 myotubes. These results suggest Myok9 cell line could be used in the development of pharmacologic treatments of skeletal muscle dysfunction or disease while remaining cognizant of lower overall protein synthesis rates.

Analysis of the Stimulative Effect of Tryptophan on Hepatic Protein Synthesis in Rats.

Tryptophan is an essential amino acid important as a protein building block, but it also serves as substrate for the generation of several bioactive compounds with important physiological roles. Furthermore, tryptophan has been reported to have a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. In the present study, the acute effects of tryptophan on protein synthesis were confirmed and compared with those of leucine in rats. Eighteen hours fasted rats were orally administered of tryptophan or leucine at a dose of 135 mg/100 g body weight by gavage and then sacrificed 1 h after administration. The effects of tryptophan and leucine on the rate of protein synthesis were evaluated by the surface sensing of translation (SUnSET) method. We also examined the ability of tryptophan to induce activation of the mTOR pathway by measuring phosphorylation of 4E-BP1 and S6K1. Oral administration of tryptophan led to a stimulation of the rate of protein synthesis concomitant with activation of mTOR pathway in the liver, but not in skeletal muscle. We also investigated the sensitivity of liver protein synthesis to tryptophan administration. The half-maximal effective doses (ED50) of tryptophan in stimulating 4E-BP1 and S6K1 phosphorylation were both about 60% of daily intake. The effect of tryptophan on hepatic protein synthesis was similar to that of leucine on muscle protein synthesis, and the sensitivity of liver protein synthesis to tryptophan administration appeared to be almost the same or slightly lower than that of muscle protein synthesis to leucine administration.

(-)-Epicatechin and its colonic metabolite hippuric acid protect against dexamethasone-induced atrophy in skeletal muscle cells

Cocoa flavanols have been shown to improve muscle function and may offer a novel approach to protect against muscle atrophy. Hippuric acid (HA) is a colonic metabolite of (-)-epicatechin (EPI), the primary bioactive compound of cocoa, and may be responsible for the associations between cocoa supplementation and muscle metabolic alterations. Accordingly, we investigated the effects of EPI and HA upon skeletal muscle morphology and metabolism within an in vitro model of muscle atrophy. Under atrophy-like conditions (24h 100μM dexamethasone (DEX)), C2C12 myotube diameter was significantly greater following co-incubation with either 25μM HA (11.19±0.39μm) or 25μM EPI (11.01±0.21μm) compared to the vehicle control (VC; 7.61±0.16μm, both P < .001). In basal and leucine-stimulated states, there was a significant reduction in myotube protein synthesis (MPS) rates following DEX treatment in VC (P=.024). Interestingly, co-incubation with EPI or HA abrogated the DEX-induced reductions in MPS rates, whereas no significant differences versus control treated myotubes (CTL) were noted. Furthermore, co-incubation with EPI or HA partially attenuated the increase in proteolysis seen in DEX-treated cells, preserving LC3 α/β II:I and caspase-3 protein expression in atrophy-like conditions. The protein content of PGC1α, ACC, and TFAM (regulators of mitochondrial function) were significantly lower in DEX-treated versus. CTL cells (all P < .050). However, co-incubation with EPI or HA was unable to prevent these DEX-induced alterations. For the first time we demonstrate that EPI and HA exert anti-atrophic effects on C2C12 myotubes, providing novel insight into the association between flavanol supplementation and favourable effects on muscle health.

Balanced Free Essential Amino Acids and Resistance Exercise Training Synergistically Improve Dexamethasone-Induced Impairments in Muscle Strength, Endurance, and Insulin Sensitivity in Mice.

Our previous study shows that an essential amino acid (EAA)-enriched diet attenuates dexamethasone (DEX)-induced declines in muscle mass and strength, as well as insulin sensitivity, but does not affect endurance. In the present study, we hypothesized that the beneficial effects will be synergized by adding resistance exercise training (RET) to EAA, and diet-free EAA would improve endurance. To test hypotheses, mice were randomized into the following four groups: control, EAA, RET, and EAA+RET. All mice except the control were subjected to DEX treatment. We evaluated the cumulative rate of myofibrillar protein synthesis (MPS) using 2H2O labeling and mass spectrometry. Neuromuscular junction (NMJ) stability, mitochondrial contents, and molecular signaling were demonstrated in skeletal muscle. Insulin sensitivity and glucose metabolism using 13C6-glucose tracing during oral glucose tolerance tests were analyzed. We found that EAA and RET synergistically improve muscle mass and/or strength, and endurance capacity, as well as insulin sensitivity, and glucose metabolism in DEX-treated muscle. These improvements are accomplished, in part, through improvements in myofibrillar protein synthesis, NMJ, fiber type preservation, and/or mitochondrial biogenesis. In conclusion, free EAA supplementation, particularly when combined with RET, can serve as an effective means that counteracts the adverse effects on muscle of DEX that are found frequently in clinical settings.

Phytochemical compound PB125 attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a model of musculoskeletal decline.

Impaired mitochondrial function and disrupted proteostasis contribute to musculoskeletal dysfunction. However, few interventions simultaneously target these two drivers to prevent musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates a transcriptional programme promoting cytoprotection, metabolism, and proteostasis. We hypothesized daily treatment with a purported Nrf2 activator, PB125, in Hartley guinea pigs, a model of musculoskeletal decline, would attenuate the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis and preserve musculoskeletal function. We treated 2- and 5-month-old male and female Hartley guinea pigs for 3 and 10months, respectively, with the phytochemical compound PB125. Longitudinal assessments of voluntary mobility were measured using Any-MazeTM open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30days of treatment. Mitochondrial oxygen consumption in soleus muscles was measured using high resolution respirometry. In both sexes, PB125 (1) increased electron transfer system capacity; (2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and (3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial and cytosolic subfractions of the soleus. These effects were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, treatment with PB125 contributed to maintenance of skeletal muscle mitochondrial respiration and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these documented effects of PB125 are also accompanied by slowed progression of other aspects of musculoskeletal dysfunction. KEY POINTS: Aside from exercise, there are no effective interventions for musculoskeletal decline, which begins in the fifth decade of life and contributes to disability and cardiometabolic diseases. Targeting both mitochondrial dysfunction and impaired protein homeostasis (proteostasis), which contribute to the age and disease process, may mitigate the progressive decline in overall musculoskeletal function (e.g. gait, strength). A potential intervention to target disease drivers is to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2) activation, which leads to the transcription of genes responsible for redox homeostasis, proteome maintenance and mitochondrial energetics. Here, we tested a purported phytochemical Nrf2 activator, PB125, to improve mitochondrial function and proteostasis in male and female Hartley guinea pigs, which are a model for musculoskeletal ageing. PB125 improved mitochondrial respiration and attenuated disease- and age-related declines in skeletal muscle protein synthesis, a component of proteostasis, in both male and female Hartley guinea pigs.

Rates of protein synthesis are maintained in brain but reduced in skeletal muscle during dietary sulfur amino acid restriction.

Dietary interventions such as sulfur amino acid restriction (SAAR) target multiple drivers of aging, and show promise for preventing or delaying the onset of chronic diseases. SAAR promotes metabolic health and longevity in laboratory animals. The effects of SAAR on proteostasis remain relatively unexplored. We previously reported that SAAR promotes mitochondrial proteostatic maintenance, despite suppression of global protein synthesis, in two peripheral tissues, the liver and skeletal muscle. However, the brain, a tissue vulnerable to age-related neurodegenerative diseases due to the loss of proteostasis, has not been thoroughly studied. Therefore, we sought to reveal proteostatic responses in the brains of mice fed SAAR for 35 days. Here, we demonstrate that male C57Bl/6J mice fed two levels of SAAR maintained rates of protein synthesis in all sub-cellular fractions of the pre-frontal cortex. In comparison, rates of skeletal muscle protein synthesis in SAAR fed mice were slower than control-fed mice. To gain mechanistic insight, we examined several key nutrient/energy sensitive signaling proteins: AMP-activated protein kinase (AMPK), eukaryotic initiation factor 2 (eIF2), and ribosomal protein S6 (rpS6). SAAR had minimal to modest effects on the total abundance and phosphorylation of these proteins in both tissues. Our results indicate that the pre-frontal cortex in brain is resistant to perturbations in protein synthesis in mice fed SAAR, unlike skeletal muscle, which had a reduction in global protein synthesis. The results from this study demonstrate that proteostatic control in brain is of higher priority than skeletal muscle during dietary SAAR.

Mechanical Disturbance of Osteoclasts Induces ATP Release That Leads to Protein Synthesis in Skeletal Muscle through an Akt-mTOR Signaling Pathway.

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X7 receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.

Improved rRNA extraction from biofouling and bioreactor samples

Mixed microbial communities are major drivers of biofouling phenomena that lead to deterioration of materials or compromise process efficiency and are also extensively employed in environmental biotechnology. The metabolically active organisms in these communities are central to further development of environmental bioprocesses or of countermeasures for biofouling control. These organisms can be targeted selectively by analysis of ribosomal RNA because the number of ribosomes in a cell is proportional to the rate of protein synthesis. Comparative analysis of the deficiencies of 6 different rRNA extraction protocols using activated sludge as model system led to the formulation of a new protocol, that consistently produced cleaner extracts from fresh or frozen samples of activated sludge with better rRNA quality and better preservation of the proportions between signatures from different community members after 10 cycles PCR. PCR amplification was best with pfu buffer + 4 mM MgCl2 at a lower annealing temperature of 60 °C. Inhibition of PCR occurred in solutions with 2% formamide. rRNA of good quality was extracted with the new protocol from samples of corrosion deposits, of both the solid and liquid fraction of chalcopyrite bioleaching experiments, of biofilms on slow sand filter media, from walls of water reservoirs and of water pipes, as well as of fouling deposits on reverse osmosis membranes.

Metabolic and molecular responses of human patellar tendon to concentric- and eccentric-type exercise in youth and older age

Exercise training can induce adaptive changes to tendon tissue both structurally and mechanically; however, the underlying compositional changes that contribute to these alterations remain uncertain in humans, particularly in the context of the ageing tendon. The aims of the present study were to determine the molecular changes with ageing in patellar tendons in humans, as well as the responses to exercise and exercise type (eccentric (ECC) and concentric (CON)) in young and old patellar tendon. Healthy younger males (age 23.5 ± 6.1 years; n = 27) and older males (age 68.5 ± 1.9 years; n = 27) undertook 8 weeks of CON or ECC training (3 times per week; at 60% of 1 repetition maximum (1RM)) or no training. Subjects consumed D2O throughout the protocol and tendon biopsies were collected after 4 and 8 weeks for measurement of fractional synthetic rates (FSR) of tendon protein synthesis and gene expression. There were increases in tendon protein synthesis following 4 weeks of CON and ECC training (P < 0.01; main effect by ANOVA), with no differences observed between young and old males, or training type. At the transcriptional level however, ECC in young adults generally induced greater responses of collagen and extracellular matrix-related genes than CON, while older individuals had reduced gene expression responses to training. Different training types did not appear to induce differential tendon responses in terms of protein synthesis, and while tendons from older adults exhibited different transcriptional responses to younger individuals, protein turnover changes with training were similar for both age groups.

The molecular basis of cereal grain proteostasis.

Storage proteins deposited in the endosperm of cereal grains are both a nitrogen reserve for seed germination and seedling growth and a primary protein source for human nutrition. Detailed surveys of the patterns of storage protein accumulation in cereal grains during grain development have been undertaken, but an in-depth understanding of the molecular mechanisms that regulate these patterns is still lacking. Accumulation of storage proteins in cereal grains involves a series of subcellular compartments, a set of energy-dependent events that compete with other cellular processes, and a balance of protein synthesis and protein degradation rates at different times during the developmental process. In this review, we focus on the importance of rates in cereal grain storage protein accumulation during grain development and outline the potential implications and applications of this information to accelerate modern agriculture breeding programmes and optimize energy use efficiency in proteostasis.

Abstract P2026: S100a1 Protects Cardiomyocytes From Hypertrophic Growth By Controlling The De-novo Synthesis Of Contractile And Mitochondrial Protein Programs

Background and Hypothesis: Loss of S100A1 expression in heart failure contributes to adverse cardiomyocyte (CM) remodeling via an unknown molecular mechanism. Conversely, the antihypertrophic effect of viral-based restoration of S100A1 expression in diseased hearts is not understood yet. We hypothesized that S100A1 may directly interfere with mRNA translation in CMs. Methods and Results: An unbiased mass spectrometry/affinity purification approach using human recombinant S100A1 protein as a bait and murine S100A1 -/- knock-out CM homogenates as prey identified the translation initiation factors EIF4G, EIF2b, RPS6 and PABP as targets for Ca 2+ -bound (holo) S100A1 protein, amongst other translational regulators. Computational modeling revealed top-scoring binding modes for holo-S100A1’s C-terminus in close proximity to RPS6’s ser-235/236 phosphorylation site harboring domain, and PLA detected S100A1/RPS6 complexes in neonatal ventricular cardiomyocytes (NVCMs). siRNA silencing of S100A1 in NVCMs caused a significant increase in cell size (+44%*) and fetal gene expression (e.g. MYH7 4.2-fold*) with a prompt enhancement of the de-novo protein synthesis rate (+89%*), as assessed by IF, RT-PCR and puromycin incorp. up to 48h (n=3-5, *P&lt;0.05 vs contr). Although phospho-specific IB revealed an increase in RPS6-ser235/236 phosphorylation (+38%, n=10, P&lt;0.05) due to S100A1 knock-down, the activity of the upstream Akt/mTORC2 pathway remained unaltered. A nascent proteome analysis unveiled the significant increase and decrease in the de-novo translation of 554 and 17 proteins (FC&gt;1.5), respectively, in S100A1-silenced NVCMs vs contr. with e.g. MYH7, ALC-1, ACTN2 or Nppb in the top 10 newly synthesized proteins. Heart muscle contraction and metabolism-related GO-terms (e.g. myofibril assembly or FA oxidation and OXPHOS) characterized the top 100 upregulated proteins. Conclusion: This is the first study providing evidence for an impact of S100A1 on the mRNA translation machinery in CMs by inhibiting translation of contractile and mitochondrial protein programs required for hypertrophic growth. S100A1 may mechanistically interfere with molecular factors of the translation initiation machinery, which warrants further investigation.

A protein synthesis brake for hematopoietic stem cell maintenance.

Bmi1 is essential for normal and leukemic hematopoiesis, but its target genes in hematopoietic stem cells (HSCs) are incompletely understood. In this issue of Genes & Development, Burgess et al. (pp. 887-900) demonstrate a novel role of Bmi1 in regulating ribosome biogenesis and protein synthesis. Bmi1-deficient HSCs exhibited reduced transplantability, with the up-regulation of ARX and genes involved in ribosome biogenesis. However, depletion of ARX or its known targets, p16 Ink4a /p19 Arf , only partially rescues Bmi1 loss-induced hematopoietic defects. They further demonstrate an increased protein synthesis rate and resultant proteostatic stress in Bmi1 -/- HSCs, indicating a novel mechanism by which Bmi1 controls HSC maintenance.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

Orchestrated translation specializes dinoflagellate metabolism three times per day

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BackgroundIGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells.MethodsL6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling.ResultsHigh AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, p<0.05) and increased L-[U-14C]-phenylalanine incorporation compared to Low AA in confluent, non-proliferating muscle cells. Blocking IGF-1 signaling by chemical inhibitors reduced IGF-1 mRNA upregulation (~50%, p< 0.01), without decrease of protein synthesis. SiRNA knockdown of IGF-1 reduced protein synthesis, mainly explained by reduced cell proliferation. High AA or IGF-1 inhibitors did not change IGF-1 receptor mRNA expressions.ConclusionAmino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids.

Review: Influence of postabsorptive metabolism on essential amino acid partitioning in lactating dairy cows

In the lactating cow, essential amino acids (EAAs) absorbed from the gut are partitioned to mammary and extra-mammary tissues via blood plasma circulation. There is also entry of EAA into plasma from the breakdown of proteins in the cow’s body. A balance model across plasma was solved to integrate entry rates of branched-chain (BCAA) and non-branched-chain EAA (NBAA) with their corresponding rate constants for clearance by mammary glands and the remainder of the body, for selected glucose and fat infusion experiments. Endogenous EAA entry from whole-body proteolysis was reduced by glucose and unchanged or increased by fat, the efficiency of net plasma BCAA clearance by mammary and extra-mammary tissues was elevated by glucose but slightly reduced by fat, and the efficiency of extra-mammary NBAA clearance may have decreased during glucose infusion but it was not affected or slightly increased by fat. These differences between glucose and fat responses can be accounted for by insulin and glucagon. Insulin suppresses endogenous EAA entry through mechanistic target of rapamycin complex 1, integrated stress response, and glycogen synthase kinase 3 signaling networks in skeletal muscle. While these networks can also regulate protein synthesis rates in muscle and the extra-mammary body, they exhibit low sensitivities to insulin in lactating ruminants. However, in the mammary glands, via these same networks, insulin stimulates clearance of EAA from plasma, although the drive to maintain a set point for milk protein yield takes precedence over nutritional signals. The glucose-induced increase in mammary BCAA clearance without an effect on NBAA clearance is due to a pronounced decrease in plasma BCAA concentrations. Because NBAAs do not experience a similar decline in concentration, the BCAA effect must be due to their metabolic transformation as opposed to sequestration in proteins. In adipose, the products of BCAA catabolism are lipogenic precursors. We propose that faster lipogenesis in adipose tissue, stimulated by glucose infusion, also promotes the uptake of precursor BCAA from plasma, causing a drop in their circulating concentrations. In addition, insulin stimulates BCAA oxidation in muscle as an alternative fuel to fatty acids. A lower efficiency of extra-mammary NBAA clearance during glucose infusion may be the consequence of decreased hepatic expression of AA-catabolizing enzymes in response to low glucagon concentration. The proportion of EAA entry partitioned to the mammary glands is a culmination of regulatory shifts at all of the points discussed above according to a regulated or unfair competition between mammary and extra-mammary processes.

Confirmation of Decreased Rates of Cerebral Protein Synthesis In Vivo in a Mouse Model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that results in intellectual disability and, in ∼50% of patients, autism spectrum disorder. The protein products that are altered in TSC (TSC1 and TSC2) form a complex to inhibit the mammalian target of rapamycin [mTOR; mTOR complex 1 (mTORC1)] pathway. This pathway has been shown to affect the process of mRNA translation through its action on ribosomal protein S6 and 4-elongation binding protein 1. It is thought that mutations in the TSC proteins lead to upregulation of the mTORC1 pathway and consequently an increase in protein synthesis. Unexpectedly, our previous study of a mouse model of TSC (Tsc2Djk+/−) demonstrated decreased in vivo rates of protein synthesis throughout the brain. In the present study, we confirm those results in another Tsc2+/− mouse model, one with a different mutation locus and on a mixed background (Tsc2Mjg+/−). We also examine mTORC1 signaling and possible effects of prior isoflurane anesthesia. Because measurements of protein synthesis rates in vivo require surgical preparation of the animal and anesthesia, we examine mTORC1 signaling pathways both under baseline conditions and following recovery from anesthesia. Our results demonstrate regionally selective effects of prior anesthesia. Overall, our results in both in vivo models suggest divergences from the central hypothesis regarding TSC and show the importance of studying protein synthesis in vivo.

Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.

Identification of autophagic protein cargo in plants in autophagy-related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific glycolytic enzymes and of other cytosol, chloroplast, and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Protein localization in transformed protoplasts and degradation assays in the presence of inhibitors confirm a role for autophagy in degrading glycolytic enzymes. Autophagy induction by phosphate (Pi) limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5-associated protein cargo of low Pi-induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism.

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.

Protein metabolism, body composition and oxygen consumption in young bulls divergent in residual feed intake offered two contrasting forage-based diets

Protein metabolism and body composition have been identified as major determinants of residual feed intake (RFI) in beef cattle fed high-starch fattening diets. This study aimed to evaluate if these two identified RFI determinants in beef cattle are the same across two contrasting silage-based diets. During two consecutive years, an 84-day feed efficiency test (Test A) immediately followed by a second 112-day feed efficiency test (Test B) was carried out using a total of 100 animals offered either one of two diets (either corn silage- or grass silage-based) over 196 days. At the end of Test A, the 32 animals most divergent for RFI (16 extreme RFI animals per diet, eight low RFI and eight high RFI) were identified and evaluated during Test B for their i) N use efficiency (NUE; N retention/N intake) calculated either from a 10-d nitrogen balance trial or from estimations based on body composition changes occurring during the whole experiment (Test A and Test B; 196 days), ii) carcass and whole-body protein turnover rates analysed through the 3-methyl-histidine urinary excretion and the N isotopic turnover rates of urine, respectively, and iii) body composition measured at the slaughterhouse at the end of Test B. Oxygen consumption was measured during Test B for the 100 animals by two GreenFeed systems. Irrespective of the diet, efficient RFI animals tended (P = 0.08) to improve their NUE when N retention was estimated for 196 days or when considering their lower urinary urea-N to total N ratio (P = 0.03). In contrast, NUE calculated during the 10-d N balance showed no differences (P = 0.65) across RFI groups suggesting that this method may not be suitable to capture small NUE differences. Efficient RFI individuals presented higher dressing percentage and muscle deposition in the carcass (P = 0.003) but lighter rumen (P = 0.001), and a trend for lower oxygen consumption (P = 0.08) than inefficient RFI animals irrespective of the diet. Lower protein degradation rates of skeletal muscle and lower protein synthesis rates of plasma proteins were found in efficient RFI cattle but only with the corn silage-based diet (RFI × Diet; P = 0.02). The higher insulinaemia associated with the corn silage-based diet (P = 0.001) seemed to be a key metabolic feature explaining the positive association between protein turnover and RFI only in this diet. Feed N was more efficiently used for growth by efficient RFI animals regardless of the diet but lower protein turnover rates in efficient RFI animals were only observed with corn silage-based diets.