Fractional Protein Synthesis Research Articles

PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets

Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P < 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P < 0.05). The phosphorylation of Akt was increased (P < 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P < 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P < 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P < 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P < 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P < 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.

Effects of gastric bypass on the digestibility and postprandial metabolic fate of 15N dietary protein in rats.

Roux-en-Y Gastric Bypass may be associated with an alteration of protein bioavailability in relation to intestinal remodeling. Our study aimed to test this hypothesis by Roux-en-Y Gastric Bypass. Diet-induced obese rats underwent Roux-en-Y Gastric Bypass surgery (RYGB rats) while a Sham-operated control group was used. All rats received a 15N-labeled protein meal 1 or 3 months after surgery and were euthanized 6h later. Protein digestibility, 15N recovered in organs and urea pool, fractional protein synthesis rate, and intestinal morphometry were assessed. Protein digestibility was similar in all groups (94.2±0.3%). The small intestine was hypertrophied in RYGB rats 1 month after surgery, weighing 9.1±0.2g vs. 7.0±0.3g in Sham rats (P = 0.003). Villus height and crypt depth were increased in the alimentary limb and ileum of RYGB rats. However, Roux-en-Y Gastric Bypass had no impact on the fractional synthesis rate. In the gastrointestinal tract, 15N retention only differed in the ileal mucosa and was higher in RYGB rats at 1 month (0.48±0.2% vs. 0.3±0.09%, P = 0.03). 15N recovery from the liver, muscle, and skin was lower in RYGB rats at 1 month. 15N recovery from urinary and plasma urea was higher in RYGB rats at both times, resulting in increased total deamination (13.2±0.9% vs. 10.1±0.5%, P<0.01). This study showed that Roux-en-Y Gastric Bypass did not affect protein digestibility. Dietary nitrogen sequestration was transitorily and moderately diminished in several organs. This was associated with a sustained elevation of postprandial deamination after Roux-en-Y Gastric Bypass, whose mechanisms merit further studies.

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Vitamin D deficiency contributes to overtraining syndrome in excessive trained C57BL/6 mice.

Overtraining syndrome is a condition resulting from excessive training load associated with inadequate recovery and poor sleep quality, leading to performance decrements and fatigue. Here we hypothesized that vitamin D (VitD) deficiency is a lead factor in the development of the overtraining syndrome. To test this hypothesis, two groups of 60-week-old C57BL/6 mice followed a 16-week excessive eccentric-based overtraining by excessive downhill running with or without dietary VitD depletion (EX and EX-D- groups). Two control groups were trained by uphill running at the same load with or without VitD depletion (CX and CX-D- groups). Handgrip strength decreased throughout the protocol for all groups but the decrease was sharper in EX-D- group (VitD × training, p = 0.0427). At the end of the protocol, the mass of Triceps brachii muscle, which is heavily stressed by eccentric contractions, was reduced in eccentric-trained groups (training effect, p = 0.0107). This atrophy was associated with a lower concentration of the anabolic myokine IL-15 (training effect, p = 0.0314) and a tendency to a higher expression of the atrogene cathepsin-L (training effect, p = 0.0628). VitD depletion led to a 50% decrease of the fractional protein synthesis rate in this muscle (VitD effect, p = 0.0004) as well as decreased FGF21 (VitD effect, p = 0.0351) and increased osteocrin (VitD effect, p = 0.038) concentrations that would lead to metabolic defects. Moreover, the proportion of anti-inflammatory Th2 lymphocytes was significantly decreased by the combination of eccentric training with VitD depletion (vitD × training, p = 0.0249) suggesting a systemic inflammation. Finally, exploratory behavior time of mice was decreased by VitD depletion (VitD effect, p = 0.0146) suggesting a cognitive dysfunction. Our results suggest that VitD deficiency exacerbates the effects of overtraining.

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

Regulation of skeletal muscle protein synthesis in the preterm pig by intermittent leucine pulses during continuous parenteral feeding.

Extrauterine growth restriction is a common complication of preterm birth. Leucine (Leu) is an agonist for the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway that regulates translation initiation and protein synthesis in skeletal muscle. Previously, we showed that intermittent intravenous pulses of Leu to neonatal pigs born at term receiving continuous enteral nutrition increases muscle protein synthesis and lean mass accretion. Our objective was to determine the impact of intermittent intravenous pulses of Leu on muscle protein anabolism in preterm neonatal pigs administered continuous parenteral nutrition. Following preterm delivery (on day 105 of 115 gestation), pigs were fitted with umbilical artery and jugular vein catheters and provided continuous parenteral nutrition. Four days after birth, pigs were assigned to receive intermittent Leu (1600 µmol kg-1 h-1 ; n = 8) or alanine (1600 µmol kg-1 h-1 ; n = 8) parenteral pulses every 4 h for 28 h. Anabolic signaling and fractional protein synthesis were determined in skeletal muscle. Leu concentration in the longissimus dorsi and gastrocnemius muscles increased in the leucine (LEU) group compared with the alanine (ALA) group (P < 0.0001). Despite the Leu-induced disruption of the Sestrin2·GATOR2 complex, which inhibits mTORC1 activation, in these muscles (P < 0.01), the abundance of mTOR·RagA and mTOR·RagC was not different. Accordingly, mTORC1-dependent activation of 4EBP1, S6K1, eIF4E·eIF4G, and protein synthesis were not different in any muscle between the LEU and ALA groups. Intermittent pulses of Leu do not enhance muscle protein anabolism in preterm pigs supplied continuous parenteral nutrition.

Prematurity Attenuates Skeletal Muscle Anabolism of Neonatal Pigs Independently of Birth Weight

ObjectivesPremature infants are at risk for postnatal growth failure that reduces lifelong lean mass. We have shown previously that preterm birth blunts the feeding-induced activation of the mechanistic target of rapamycin (mTOR) signaling pathway, translation initiation, and protein synthesis in skeletal muscle of neonatal pigs. Considering that body weight can vary widely at birth, we sought to separate the effect of prematurity from birth weight on the anabolic response to feeding. MethodsPigs were delivered by cesarean section at preterm (103 d) or term (112 d) gestation and fed parenterally for 4 d. On day 4, pigs were fasted for 4 h and were either fasted one additional hour or fed by oral gavage, yielding four groups: 1) preterm fasted (PT-FAST); 2) preterm fed (PT-FED); 3) term fasted (T-FAST); and 4) term fed (T-FED). All pigs were injected with a flooding dose of L-[4-3H]Phe to determine fractional protein synthesis rates and euthanized 60 min after feeding or the additional fast. Pigs were stratified by birth weight such that the highest birth weight preterm pigs were compared with the lowest birth weight term pigs (n = 10–13 per group). ResultsDespite similar birth weight in preterm and term pigs (1035 vs 993 ± 54 g, P > 0.10), relative weight gain of preterm was 33% lower than term pigs (22.0 vs. 32.9 ± 1.6 g·kg–1·d–1, P < 0.001). Longissimus dorsi (LD), gastrocnemius, and diaphragm protein synthesis rates were not different between PT-FAST and T-FAST groups. Gastrocnemius protein synthesis was 18% lower in PT-FED than T-FED group (P < 0.01), but protein synthesis was not different between PT-FED and T-FED groups in LD and diaphragm muscles. The abundance of the active eIF4E·eIF4G complex and phosphorylation of 4E-BP1 and S6K1, readouts of mTOR activation and translation initiation signaling, were increased after feeding in all muscles (P < 0.001). Translation initiation signaling was lower in LD and gastrocnemius muscles in PT-FED than T-FED groups (P < 0.05) but was not different in diaphragm muscle (P > 0.10). ConclusionsThese findings imply that prematurity, independent of birth weight, blunts mTOR activation and protein synthesis after feeding. The blunted anabolic response to feeding likely underpins postnatal growth failure often observed in infants born preterm. Funding SourcesNIH and USDA.

HuR-dependent SOD2 protein synthesis is an early adaptation to anchorage-independence

During metastasis cancer cells must adapt to survive loss of anchorage and evade anoikis. An important pro-survival adaptation is the ability of metastatic tumor cells to increase their antioxidant capacity and restore cellular redox balance. Although much is known about the transcriptional regulation of antioxidant enzymes in response to stress, how cells acutely adapt to alter antioxidant enzyme levels is less well understood. Using ovarian cancer cells as a model, we demonstrate that an increase in mitochondrial superoxide dismutase SOD2 protein expression is a very early event initiated in response to detachment, an important step during metastasis that has been associated with increased oxidative stress. SOD2 protein synthesis is rapidly induced within 0.5–2 h of matrix detachment, and polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 mRNA translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1 and promotes HuR binding to SOD2 mRNA. Using HuR siRNA-mediated knockdown, we show that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and consequent nascent SOD2 protein synthesis in anchorage-independence. Cellular detachment also activates the stress-response mitogen-activated kinase p38, which is necessary for HuR-SOD2 mRNA interactions and induction of SOD2 protein output. These findings illustrate a novel translational regulatory mechanism of SOD2 by which ovarian cancer cells rapidly increase their mitochondrial antioxidant capacity as an acute stress response to anchorage-independence.

Moderate adiposity levels counteract protein metabolism modifications associated with aging in rats

Physiological parameters such as adiposity and age are likely to influence protein digestion and utilization. The aim of this study was to evaluate the combined effects of age and adiposity on casein protein and amino acid true digestibility and its postprandial utilization in rats. Four groups were included (n = 7/8): 2months/normal adiposity, 2months/high adiposity, 11months/normal adiposity and 11months/high adiposity. Rats were given a calibrated meal containing 15N-labeled casein (Ingredia, Arras, France) and were euthanized 6h later. Digestive contents were collected to assess protein and amino acid digestibilities. 15N enrichments were measured in plasma and urine to determine total body deamination. Fractional protein synthesis rate (FSR) was determined in different organs using a flooding dose of 13C valine. Nitrogen and amino acid true digestibility of casein was around 95-96% depending on the group and was increased by 1% in high adiposity rats (P = 0.04). Higher adiposity levels counteracted the increase in total body deamination (P = 0.03) that was associated with older age. Significant effects of age (P = 0.006) and adiposity (P = 0.002) were observed in the muscle FSR, with age decreasing it and adiposity increasing it. This study revealed that a higher level of adiposity resulted in a slight increase in protein and individual amino acid true digestibility values and seemed to compensate for the metabolic postprandial protein alterations observed at older age.

337 Efficient RFI Bulls Presented Lower Fractional Synthesis Rates of Plasma Proteins When Fed Corn but Not Grass Based Diets

Abstract Protein turnover (PT), the continual synthesis and degradation of body proteins not leading to protein gain, is an essential high energy-demanding process. We assumed that PT might explain the between-animal variations of residual feed intake (RFI). The objective was to measure PT in extreme RFI cattle fed two contrasted diets (grass or corn-based). We conducted a RFI test for 84 days with 100 Charolais bulls and we selected the 32 most extreme (8 per diet and RFI group) for PT measurements using 1) the urinary 3-methyl-histidine to creatinine ratio, as a biomarker of the fractional protein degradation rate (FDR) of skeletal-muscle and 2) the isotopic N turnover rate measured in urine and plasma, as a proxy, respectively, of the whole-body FDR and the fractional protein synthesis rates (FSR) of plasma proteins. The 3-methyl-histidine and creatinine were determined from 10 d total urine collection. Isotopic N turnover in urine and plasma was evaluated by modelling the 15N depletion rate over 112 d following an isotopic N dietary change. Higher plasma FSR and higher skeletal-muscle and whole-body FDR were observed with corn-vs-grass diets (≥11%; P ≤ 0.03), in line with higher metabolizable protein and net energy intakes (≥10%, P = 0.001). Differences between extreme RFI animals were noted with the corn diets only, where efficient animals presented significant lower plasma FSR (-10%; P = 0.04) and numerically lower skeletal-muscle and whole-body FDR (-13% and - 8.9%; P &amp;gt; 0.16 respectively) than non-efficient. Non-significant differences were probably due to an insufficient size of our experimental setup. Plasma FSR is related to the PS of hepatic exportation, hence the lower plasma FSR observed in efficient RFI animals fed corn diets may reflect a lower organs to carcass ratio. Altogether results suggests that efficient RFI bulls fed corn diets had a lower hepatic PT with no-significant changes of whole-body and skeletal muscle PT.

Mammary Development in Gilts at One Week Postnatal Is Related to Plasma Lysine Concentration at 24 h after Birth, but Not Colostrum Dose.

Simple SummaryA relationship exists between a female’s early nutritional environment and her ability to produce milk when she lactates as an adult. Colostrum is the first milk available to neonates after birth. We hypothesized that differing levels of colostrum stimulate differences in very early mammary development. Despite differences in weight at 24 h and 7 days, mammary morphological development and DNA content was not found to be different between gilts fed a high versus low dose of colostrum. The rate of mammary gland protein and DNA synthesis over the first week was not different between the groups. Circulating levels of amino acids were determined after 24 h of colostrum feeding, and levels of circulating lysine were found to be related to average daily gain and mammary DNA synthetic rate. Moreover, the level of lysine was related to a lower ratio of DNA to protein synthesis, suggesting that higher lysine favored cell division versus differentiation (by leaving the cell cycle). Further studies are needed in this area.Perinatal nutrition affects future milk production. The number of mammary epithelial cells affect milk production capacity. Therefore, it was hypothesized that the level of colostrum intake affects the proliferation rate and the total number of mammary epithelial cells in the gland. The ratio of newly synthesized protein to newly synthesized DNA reflects the relative amount of cellular differentiation to cell division. The study objective was to determine the relationship between the level of colostrum intake and 24 h-level of circulating amino acid, glucose and insulin with mammary parenchyma histological features, cell division and protein synthesis over the first week postnatal. One of two standardized doses of a homogenate colostrum sample, 10% (n = 8) and 20% (n = 8) of birth bodyweight, was fed to gilts over the first 24 h postnatal. Gilts were administered deuterium oxide immediately after birth and daily to label newly synthesized DNA and proteins. Gilts were euthanized on postnatal day seven, and DNA and protein were isolated from mammary parenchyma. DNA and protein fractional synthesis (f) and fractional synthetic rate (FSR) were calculated using mass isotopomer distribution analysis. The ratio of protein f and FSR to DNA f and FSR were calculated and used to indicate the relative amounts of differentiation to cell division. Mammary morphological development was also analyzed by measuring the parenchymal epithelial area and the stromal and epithelial proliferation index on postnatal day seven. Colostrum dose was not related to any of the variables used to evaluate mammary development. However, plasma lysine levels at 24 h postnatal were positively related to average daily gain (ADG; r = 0.54, p = 0.05), DNA f (r = 0.57; p = 0.03) and DNA FSR (r = 0.57; p = 0.03) in mammary parenchyma. Plasma lysine was inversely related to the ratio of protein to DNA f and FSR (r = −0.56; p = 0.04). ADG was related to the parenchymal epithelial area and DNA and protein f and FSR (p < 0.05). These relationships support the idea that the nutritional environment affects early mammary development and that higher lysine levels in the perinatal period favored a greater degree of cell division versus differentiation in mammary of neonatal pigs and thus, warrant further investigations.

Evolution of viscera and muscle fractional protein synthesis rate in lean meat selected hybrids and castrated Duroc pigs fed under moderate crude protein restriction

Differences in producing performance and organoleptic meat characteristics among pig genotypes and/or producing types are widely known. These parameters are also subjected to the animal’s development, feeding and management. Detailed knowledge of the effects of production phase (PP), pig producing type (PT), dietary protein availability and their interactions on nutrient digestibility, nitrogen balance and protein metabolism is essential information to improve precision feeding techniques. The experiment was a 2 (PP) × 2 (PT) × 2 (diet) factorial design conducted with 32 male pigs, 16 entire F2 pigs progeny of Pietrain sires and Duroc × Landrace dams, and 16 castrated purebred Durocs belonging to two production phases (growing: 29.5 ± 3.19 v. fattening: 88.6 ± 6.26 kg BW), and assigned to one of two dietary CP levels, either standard (SP: 17% in growing and 15% in fattening) or low (LP: 15% in growing and 13% in fattening). Viscera and muscle fractional protein synthesis rates (FSRs; %/day) were conducted through a single infusion of 15% L-[ring-2H5]-phenylalanine, with subsequent blood sampling from 12 to 40 min, and sample collection of liver, duodenum, biceps femoris and longissimus dorsi skeletal muscles after sacrifice. Fattening animals acquired a greater feed ingestion capacity, average daily gain (P < 0.01) and apparent ileal digestibility, whereas growing pigs showed higher FSRs in both viscera (duodenum and liver) and in longissimus dorsi. F2 pigs showed higher average daily gain, nitrogen retention rates and FSR in liver and longissimus dorsi (P < 0.01). Nevertheless, apparent ileal digestibility in all essential amino acids was lower in F2 compared with Duroc pigs (P < 0.05). Protein metabolism was barely influenced by dietary CP content, although animals fed LP registered the lowest apparent ileal digestibility for CP and also for most of the essential amino acids compared with SP-fed pigs. This information may reveal differences in amino acid requirements between both PTs, with Duroc pigs receiving excess of dietary amino acids.

Basal protein synthesis rates differ between vastus lateralis and rectus abdominis muscle.

Background In vivo muscle protein synthesis rates are typically assessed by measuring the incorporation rate of stable isotope labelled amino acids in skeletal muscle tissue collected from vastus lateralis muscle. It remains to be established whether muscle protein synthesis rates in the vastus lateralis are representative of muscle protein synthesis rates of other muscle groups. We hypothesized that post‐absorptive muscle protein synthesis rates differ between vastus lateralis and rectus abdominis, pectoralis major, or temporalis muscle in vivo in humans.MethodsTwenty‐four patients (62 ± 3 years, 42% female), scheduled to undergo surgery, participated in this study and underwent primed continuous intravenous infusions with l‐[ring‐13C6]‐phenylalanine. During the surgical procedures, serum samples were collected, and muscle tissue was obtained from the vastus lateralis as well as from the rectus abdominis, pectoralis major, or temporalis muscle. Fractional mixed muscle protein synthesis rates (%/h) were assessed by measuring the incorporation of l‐[ring‐13C6]‐phenylalanine into muscle tissue protein.ResultsSerum l‐[ring‐13C6]‐phenylalanine enrichments did not change throughout the infusion period. Post‐absorptive muscle protein synthesis rates calculated based upon serum l‐[ring‐13C6]‐phenylalanine enrichments did not differ between vastus lateralis and rectus abdominis (0.032 ± 0.004 vs. 0.038 ± 0.003%/h), vastus lateralis and pectoralis major, (0.025 ± 0.003 vs. 0.022 ± 0.005%/h) or vastus lateralis and temporalis (0.047 ± 0.005 vs. 0.043 ± 0.005%/h) muscle, respectively (P > 0.05). When fractional muscle protein synthesis rates were calculated based upon tissue‐free l‐[ring‐13C6]‐phenylalanine enrichments as the preferred precursor pool, muscle protein synthesis rates were significantly higher in rectus abdominis (0.089 ± 0.008%/h) compared with vastus lateralis (0.054 ± 0.005%/h) muscle (P < 0.01). No differences were observed between fractional muscle protein synthesis rates in vastus lateralis and pectoralis major (0.046 ± 0.003 vs. 0.041 ± 0.008%/h) or vastus lateralis and temporalis (0.073 ± 0.008 vs. 0.083 ± 0.011%/h) muscle, respectively.ConclusionsPost‐absorptive muscle protein synthesis rates are higher in rectus abdominis when compared with vastus lateralis muscle. Post‐absorptive muscle protein synthesis rates do not differ between vastus lateralis and pectoralis major or temporalis muscle. Protein synthesis rates in muscle tissue samples obtained during surgery do not necessarily represent a good proxy for appendicular skeletal muscle protein synthesis rates.

Female mice may have exacerbated catabolic signalling response compared to male mice during development and progression of disuse atrophy.

BackgroundMuscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well‐established model of hindlimb unloading (HU).MethodsOne hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism).ResultsBoth males and females lost ~30% of tibialis anterior cross‐sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross‐sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24–48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4‐fold greater content in females compared with ~2‐fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes.ConclusionsThese results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.

Ammonium chloride administration prior to exercise has muscle-specific effects on mitochondrial and myofibrillar protein synthesis in rats.

AimExercise is able to increase both muscle protein synthesis and mitochondrial biogenesis. However, acidosis, which can occur in pathological states as well as during high‐intensity exercise, can decrease mitochondrial function, whilst its impact on muscle protein synthesis is disputed. Thus, the aim of this study was to determine the effect of a mild physiological decrease in pH, by administration of ammonium chloride, on myofibrillar and mitochondrial protein synthesis, as well as associated molecular signaling events.MethodsMale Wistar rats were given either a placebo or ammonium chloride prior to a short interval training session. Rats were killed before exercise, immediately after exercise, or 3 h after exercise.ResultsMyofibrillar (p = 0.036) fractional protein synthesis rates was increased immediately after exercise in the soleus muscle of the placebo group, but this effect was absent in the ammonium chloride group. However, in the gastrocnemius muscle NH4Cl increased myofibrillar (p = 0.044) and mitochondrial protein synthesis (0 h after exercise p = 0.01; 3 h after exercise p = 0.003). This was accompanied by some small differences in protein phosphorylation and mRNA expression.ConclusionThis study found ammonium chloride administration immediately prior to a single session of exercise in rats had differing effects on mitochondrial and myofibrillar protein synthesis rates in soleus (type I) and gastrocnemius (type II) muscle in rats.

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Prematurity blunts the insulin- and amino acid-induced stimulation of translation initiation and protein synthesis in skeletal muscle of neonatal pigs.

Extrauterine growth restriction in premature infants is largely attributed to reduced lean mass accretion and is associated with long-term morbidities. Previously, we demonstrated that prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in skeletal muscle of neonatal pigs. The objective of the current study was to determine whether the blunted feeding response is mediated by reduced responsiveness to insulin, amino acids, or both. Pigs delivered by cesarean section preterm (PT; 103 days, n = 25) or at term (T; 112 days, n = 26) were subject to euinsulinemic-euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps four days after delivery. Indices of mechanistic target of rapamycin complex 1 (mTORC1) signaling and fractional protein synthesis rates were measured after 2 h. Although longissimus dorsi (LD) muscle protein synthesis increased in response to both INS and AA, the increase was 28% lower in PT than in T. Upstream of mTORC1, Akt phosphorylation, an index of insulin signaling, was increased with INS but was 40% less in PT than in T. The abundances of mTOR·RagA and mTOR·RagC, indices of amino acid signaling, increased with AA but were 25% less in PT than in T. Downstream of mTORC1, eIF4E·eIF4G abundance was increased by both INS and AA but attenuated by prematurity. These results suggest that preterm birth blunts both insulin- and amino acid-induced activation of mTORC1 and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the high prevalence of extrauterine growth restriction in prematurity.NEW & NOTEWORTHY Extrauterine growth faltering is a major complication of premature birth, but the underlying cause is poorly understood. Our results demonstrate that preterm birth blunts both the insulin-and amino acid-induced activation of mTORC1-dependent translation initiation and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced accretion of lean mass and extrauterine growth restriction of premature infants.

Intermittent Bolus Feeding Enhances Organ Growth More Than Continuous Feeding in a Neonatal Piglet Model.

ABSTRACTBackgroundOrogastric tube feeding is frequently prescribed for neonates who cannot ingest food normally. In a piglet model of the neonate, greater skeletal muscle growth is sustained by upregulation of translation initiation signaling when nutrition is delivered by intermittent bolus meals, rather than continuously.ObjectivesThe objective of this study was to determine the long-term effects of feeding frequency on organ growth and the mechanism by which feeding frequency modulates protein anabolism in these organs.MethodsEighteen neonatal pigs were fed by gastrostomy tube the same amount of a sow milk replacer either by continuous infusion (CON) or on an intermittent bolus schedule (INT). After 21 d of feeding, the pigs were killed without interruption of feeding (CON; n = 6) or immediately before (INT-0; n = 6) or 60 min after (INT-60; n = 6) a meal, and fractional protein synthesis rates and activation indexes of signaling pathways that regulate translation initiation were measured in the heart, jejunum, ileum, kidneys, and liver.ResultsCompared with continuous feeding, intermittent feeding stimulated the growth of the liver (+64%), jejunum (+48%), ileum (+40%), heart (+64%), and kidney (+56%). The increases in heart, kidney, jejunum, and ileum masses were proportional to whole body lean weight gain, but liver weight gain was greater in the INT-60 than the CON, and intermediate for the INT-0 group. For the liver and ileum, but not the heart, kidney, and jejunum, INT-60 compared with CON pigs had greater fractional protein synthesis rates (22% and 48%, respectively) and was accompanied by an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 phosphorylation.ConclusionsThese results suggest that intermittent bolus compared with continuous orogastric feeding enhances organ growth and that in the ileum and liver, intermittent feeding enhances protein synthesis by stimulating translation initiation.

The Dunkin Hartley Guinea Pig Is a Model of Primary Osteoarthritis That Also Exhibits Early Onset Myofiber Remodeling That Resembles Human Musculoskeletal Aging.

Skeletal muscle dysfunction, articular cartilage degeneration, and bone loss occur essentially in parallel during aging. Mechanisms contributing to this systemic musculoskeletal decline remain incompletely understood, limiting progress toward developing effective therapeutics. Because the progression of human musculoskeletal aging is slow, researchers rely on rodent models to identify mechanisms and test interventions. The Dunkin Hartley guinea pig is an outbred strain that begins developing primary osteoarthritis by 4 months of age with a progression and pathology similar to aging humans. The purpose of this study was to determine if skeletal muscle remodeling during the progression of osteoarthritis in these guinea pigs resembles musculoskeletal aging in humans. We compared Dunkin Hartley guinea pigs to Strain 13 guinea pigs, which develop osteoarthritis much later in the lifespan. We measured myofiber type and size, muscle density, and long-term fractional protein synthesis rates of the gastrocnemius and soleus muscles in 5, 9, and 15-month-old guinea pigs. There was an age-related decline in skeletal muscle density, a greater proportion of smaller myofibers, and a decline in type II concomitant with a rise in type I myofibers in the gastrocnemius muscles from Dunkin Hartley guinea pigs only. These changes were accompanied by age-related declines in myofibrillar and mitochondrial protein synthesis in the gastrocnemius and soleus. Collectively, these findings suggest Dunkin Hartley guinea pigs experience myofiber remodeling alongside the progression of osteoarthritis, consistent with human musculoskeletal aging. Thus, Dunkin Hartley guinea pigs may be a model to advance discovery and therapeutic development for human musculoskeletal aging.

Elevated plasma lactate levels via exogenous lactate infusion do not alter resistance exercise-induced signaling or protein synthesis in human skeletal muscle.

Lactate has been implicated as a potential signaling molecule. In myotubes, lactate incubation increases mechanistic target of rapamycin complex 1 (mTORC1)- and ERK-signaling and induces hypertrophy, indicating that lactate could be a mediator of muscle adaptations to resistance exercise. However, the potential signaling properties of lactate, at rest or with exercise, have not been explored in human tissue. In a crossover design study, 8 men and 8 women performed one-legged resistance exercise while receiving venous infusion of saline or sodium lactate. Blood was sampled repeatedly, and muscle biopsies were collected at rest and at 0, 90, and 180 min and 24 h after exercise. The primary outcomes examined were intracellular signaling, fractional protein synthesis rate (FSR), and blood/muscle levels of lactate and pH. Postexercise blood lactate concentrations were 130% higher in the Lactate trial (3.0 vs. 7.0 mmol/L, P < 0.001), whereas muscle levels were only marginally higher (27 vs. 32 mmol/kg dry wt, P = 0.003) compared with the Saline trial. Postexercise blood pH was higher in the Lactate trial (7.34 vs. 7.44, P < 0.001), with no differences in intramuscular pH. Exercise increased the phosphorylation of mTORS2448 (∼40%), S6K1T389 (∼3-fold), and p44T202/T204 (∼80%) during recovery, without any differences between trials. FSR over the 24-h recovery period did not differ between the Saline (0.067%/h) and Lactate (0.062%/h) trials. This study does not support the hypothesis that blood lactate levels can modulate anabolic signaling in contracted human muscle. Further in vivo research investigating the impact of exercised versus rested muscle and the role of intramuscular lactate is needed to elucidate its potential signaling properties.