Differences In Protein Turnover Research Articles

People with obesity exhibit losses in muscle proteostasis that are partly improved by exercise training.

This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance profile or turnover rate of proteins in this population. Men with (n=3) or without (n=4) obesity were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (D2 O) consumption and serial biopsies of vastus lateralis muscle. Men with obesity then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1min intervals at 100% maximum aerobic power interspersed by 1min recovery periods. The number of intervals per session progressed from 4 to 8, and during weeks 8-10 the 14-d measurement protocol was repeated. Proteomic analysis detected 352 differences (p<0.05, false discovery rate<5%) in protein abundance and 19 (p<0.05) differences in protein turnover, including components of the ubiquitin-proteasome system. HIIT altered the abundance of 53 proteins and increased the turnover rate of 22 proteins (p<0.05) and tended to benefit proteostasis by increasing muscle protein turnover rates. Obesity and insulin resistance are associated with compromised muscle proteostasis, which may be partially restored by endurance exercise.

Age-Related Susceptibility to Muscle Damage Following Mechanotherapy in Rats Recovering From Disuse Atrophy.

The inability to fully recover lost muscle mass following periods of disuse atrophy predisposes older adults to lost independence and poor quality of life. We have previously shown that mechanotherapy at a moderate load (4.5 N) enhances muscle mass recovery following atrophy in adult, but not older adult rats. We propose that elevated transverse stiffness in aged muscle inhibits the growth response to mechanotherapy and hypothesize that a higher load (7.6 N) will overcome this resistance to mechanical stimuli. F344/BN adult and older adult male rats underwent 14 days of hindlimb suspension, followed by 7 days of recovery with (RE + M) or without (RE) mechanotherapy at 7.6 N on gastrocnemius muscle. The 7.6 N load was determined by measuring transverse passive stiffness and linearly scaling up from 4.5 N. No differences in protein turnover or mean fiber cross-sectional area were observed between RE and RE + M for older adult rats or adult rats at 7.6 N. However, there was a higher number of small muscle fibers present in older adult, but not adult rats, which was explained by a 16-fold increase in the frequency of small fibers expressing embryonic myosin heavy chain. Elevated central nucleation, satellite cell abundance, and dystrophin-/laminin+ fibers were present in older adult rats only following 7.6 N, while 4.5 N did not induce damage at either age. We conclude that age is an important variable when considering load used during mechanotherapy and age-related transverse stiffness may predispose older adults to damage during the recovery period following disuse atrophy.

RAPID COMMUNICATION: Residual feed intake in beef cattle is associated with differences in protein turnover and nutrient transporters in ruminal epithelium.

Residual feed intake (RFI) is a widely used measure of feed efficiency in cattle. Although the precise biologic mechanisms associated with improved feed efficiency are not well-known, most-efficient steers (i.e., with low RFI coefficient) downregulate abundance of proteins controlling protein degradation in skeletal muscle. Whether cellular mechanisms controlling protein turnover in ruminal tissue differ by RFI classification is unknown. The aim was to investigate associations between RFI and signaling through the mechanistic target of rapamycin (MTOR) and ubiquitin-proteasome pathways in ruminal epithelium. One hundred and forty-nine Red Angus cattle were allocated to 3 contemporary groups according to sex and herd origin. Animals were offered a finishing diet for 70 d to calculate the RFI coefficient for each. Within each group, the 2 most-efficient (n = 6) and least-efficient animals (n = 6) were selected. Compared with least-efficient animals, the most-efficient animals consumed less feed (P < 0.05; 18.36 vs. 23.39 kg/d DMI). At day 70, plasma samples were collected for insulin concentration analysis. Ruminal epithelium was collected immediately after slaughter to determine abundance and phosphorylation status of 29 proteins associated with MTOR, ubiquitin-proteasome, insulin signaling, and glucose and amino acid transport. Among the proteins involved in cellular protein synthesis, most-efficient animals had lower (P ≤ 0.05) abundance of MTOR, p-MTOR, RPS6KB1, EIF2A, EEF2K, AKT1, and RPS6KB1, whereas MAPK3 tended (P = 0.07) to be lower. In contrast, abundance of p-EEF2K, p-EEF2K:EEF2K, and p-EIF2A:EIF2A in most-efficient animals was greater (P ≤ 0.05). Among proteins catalyzing steps required for protein degradation, the abundance of UBA1, NEDD4, and STUB1 was lower (P ≤ 0.05) and MDM2 tended (P = 0.06) to be lower in most-efficient cattle. Plasma insulin and ruminal epithelium insulin signaling proteins did not differ (P > 0.05) between RFI groups. However, abundance of the insulin-responsive glucose transporter SLC2A4 and the amino acid transporters SLC1A3 and SLC1A5 also was lower (P ≤ 0.05) in most-efficient cattle. Overall, the data indicate that differences in signaling mechanisms controlling protein turnover and nutrient transport in ruminal epithelium are components of feed efficiency in beef cattle.

Determinants and Regulation of Protein Turnover in Yeast.

Protein turnover maintains the recycling needs of theproteome, and its malfunction has been linked to aging and age-related diseases. However, not allproteins turnover equally, and the factors that contribute to accelerate or slow down turnover are mostly unknown. We measured turnover rates for 3,160 proteins in exponentially growing yeast and analyzed their dependence on physical, functional, and genetic properties. We found that functional characteristics, including protein localization, complex membership, and connectivity, have greater effect on turnover than sequence elements. We also found that protein turnover and mRNA turnover are correlated. Analysis under nutrient perturbation and osmotic stress revealed that protein turnover highly depends on cellular state and is faster when proteinsare being actively used. Finally, stress-induced changes in protein and transcript abundance correlated with changes in protein turnover. This study provides a resource of protein turnover rates and principles to understand the recycling needs of the proteome under basal conditions and perturbation.

Alanyl-glutamine but not glycyl-glutamine improved the proliferation of enterocytes as glutamine substitution in vitro.

The synthetic dipeptides alanyl-glutamine (Ala-Gln) and glycyl-glutamine (Gly-Gln) are used as Gln substitution to provide energy source in the gastrointestinal tract due to their high solubility and stability. This study aimed to investigate the effects of Gln, Ala-Gln and Gly-Gln on mitochondrial respiration and protein turnover of enterocytes. Intestinal porcine epithelial cells (IPEC-J2) were cultured for 2days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 2.5mM Gln, Ala-Gln or Gly-Gln. Results from 5-ethynyl-2'-deoxyuridine incorporation and flow cytometry analysis indicated that there were no differences in proliferation between free Gln and Ala-Gln-treated cells, whereas Gly-Gln treatment inhibited the cell growth compared with Gln treatment. Significantly lower mRNA expressions of Sp1 and PepT1 were also observed in Gly-Gln-treated cells than that of Ala-Gln treatment. Ala-Gln treatment increased the basal respiration and ATP production, compared with free Gln and Gly-Gln treatments. There were no differences in protein turnover between free Gln and Ala-Gln-treated cells, but Gly-Gln treatment reduced protein synthesis and increased protein degradation. Ala-Gln treatment stimulated mTOR activation whereas Gly-Gln decreased mTOR phosphorylation and increased the UB protein expression compared with free Gln treatment. These results indicate that Ala-Gln has the very similar functional profile to free Gln in porcine enterocytes in vitro and can be substituted Gln as energy and protein sources in the gastrointestinal tract.

Residual Feed Intake as a Tool for Selecting More Efficient Animals: A Review

Feeds alone contributes about 60–70% of total cost of ruminant's production. There is deficiency to the tune of 10, 37 and 35% for dry fodder, concentrates and green fodders, respectively in India. Therefore, selection of efficient animals could help in reducing the burden of already scarce feed resources. A significant improvement in profitability could be achieved through a reduction in production costs by implementation of selection strategies to improve feed efficiency. There are several measures of feed efficiency viz., feed conversion ratio, relative growth rate, partial efficiency of growth, gross efficiency and residual feed intake (RFI). RFI is difference between actual feed intake and the feed an animal is expected to consume based on its body size and growth rate. Cattle with low RFI eat less than expected for their weight and growth rate and are, therefore, more efficient than cattle with high RFI. Among the feed efficiency measures, RFI is considered to be the best as it is moderately heritable (h2=0.29–0.46) and phenotypically independent of growth rate and body weight in growing cattle and basically reflects the variation in animal's maintenance. This makes RFI as selection tool for efficient animals. About one-third of the biological variation in RFI of these calves could be explained by differences in feed intake (2%), digestion (10%), heat increment (9%), composition of gain (5%) and activity (5%) while the remaining variation in RFI was related to differences in protein turnover, stress and tissue metabolism (37%) and differences in cellular energy expenditures such as ion pumping and mitochondrial proton leakage (27%). The low RFI animals consume less feed and produce less enteric methane. hence, there are economic and environmental benefits by selecting such efficient animals. Phenotypic selection for RFI poses challenges because it requires measurement of each individual's feed intake for a minimum of 70 days which is expensive, time and labour intensive. As a result, there is need to develop alternative approaches like biochemical and physiological markers which could differentiate between high and low RFI animals within short period of time. Recently, genomic selection is being used as an appealing alternative tool for selection of low RFI animals. However, such studies need to be conducted using large number of animals so that more efficient animals could be selected for future breeding programmes for economic and environmental benefits.

Net Protein Balance after Load Carriage Exercise is Enhanced by Amino Acid Supplementation

Essential amino acid (EAA) supplementation during conventional endurance (CE) exercise attenuates post-exercise protein breakdown (PB) and enhances net protein balance (NET). Whether consuming EAA during load carriage endurance (LC) exercise confers the same anabolic effect is not clear, as the metabolic and mechanical strain of LC could upregulate protein turnover and branched-chain amino acid oxidation (OX). This study examined whole-body protein turnover during recovery from LC and CE with and without EAA supplementation using the reciprocal-pool model. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, 4.0 ± 0.5 L•min-1) were randomly assigned to perform a 90 min LC (24 ± 3 kg load), or metabolically-matched (2.2 ± 0.1 L•m-1), CE (cycle ergometry) exercise bout, during which EAA (10 g EAA, 37% leucine) or nonnutritive control (CON) drinks were consumed. Post-exercise PB was lower and OX was higher for EAA (53 ± 13 and 23 ± 4 µmol·kg-1·h-1) than CON (102 ± 13 and 10 ± 1 µmol·kg-1·h-1), irrespective of exercise mode (P < 0.05). Similarly, NET was not influenced by exercise mode, but was higher (P < 0.05) for EAA (49 ± 8 µmol·kg-1·h-1) than CON (-3 ± 1 µmol·kg-1·h-1). No other differences in protein turnover were observed. These data confirm the protein-sparing effect of EAA supplementation during endurance exercise, and suggest that if the metabolic demands are equivalent, whole-body protein turnover responses to weighted and non-weight bearing endurance exercise are the same. Supported by US Army Medical Research and Material Command and US Army Combat Feeding Directorate.

Dynamic13C-labeling experiments prove important differences in protein turnover rate between twoSaccharomyces cerevisiaestrains

We developed a method for the quantification of protein turnover rates using (13)C-labeled substrates combined with the analysis of the labeling patterns of proteinogenic amino acids. Using this method, the specific amino acid turnover rates between proteins and the pool of free amino acids were determined for eight different amino acids (alanine, valine, proline, aspartic acid, glycine, leucine, isoleucine, and threonine) in two Saccharomyces cerevisiae strains (CEN.PK 113-7D and YSBN2). Furthermore, proteasome activities were compared for both strains. Both results confirmed the hypothesis of a higher protein turnover rates in CEN.PK 113-7D, which was generated in a previous comparative systems biology study of these two yeast strains. The ATP costs associated with the observed differences in protein turnover were quantified and could explain accurately the differences in biomass yield between both strains that are observed in chemostat cultures. The percentage of maintenance ATP associated with protein polymerization (polymerization for growth and re-polymerization because of turnover) and degradation was estimated to be 72% for YSBN2 and 79% for CEN.PK 113-7D, which makes these processes the dominant nonbiosynthetic drain of ATP in living cells, and hence, it represents an energetic parameter of great relevance.

The effects of added sulphur amino acids, threonine and an ideal amino acid ratio on nitrogen metabolism in mature, overweight dogs

The objectives of this study were to investigate the effects of added essential amino acids in conjunction with a dietary lysine/MJ of 0.72 on nitrogen (N) metabolism in dogs. Treatments were; a control diet, a diet that provided an ideal amino acid profile (IAA), a diet with added total sulphur amino acids (TSAA), and a diet with added TSAA and threonine (TT). Diets were fed to eight overweight, mature, female hounds using a replicated 4 × 4 Latin Square design. Food intake was similar across treatments, however, food N intake was higher (p < 0.001) for TSAA than control, IAA or TT. Nitrogen absorbed was higher (p < 0.01) for TSAA than IAA and control. Urea N excretion was greater for control than TT (p < 0.05). Urine N excretion did not differ between diets. There were no differences in digestibility or N retention of diets. There were no differences in protein turnover, synthesis, or degradation. Blood metabolites were within normal ranges and did not differ due to dietary treatment. Based on the measurements made in this study, there is no benefit for added TSAA, TT or additional EAA in diets for mature dogs formulated to provide a 0.72 g lysine/MJ ME ratio.

Whole Body Protein Turnover and Urea Production of Preterm Small for Gestational Age Infants Fed Fortified Human Milk or Preterm Formula

To investigate protein metabolism and urea production in preterm small for gestational age neonates fed a preterm formula or fortified human milk. Ten preterm small for gestational age neonates were fed either their own mother's milk fortified with a powdered protein mineral supplement or a special preterm formula. Protein metabolism was determined using constant steady-state infusion of L-[ring-2H5]phenylalanine and L-[1-13C]valine. Urea production was determined from steady-state [13C]urea kinetics. Mean protein intake was 24% higher in the preterm formula group than in the fortified human milk group. No differences in protein turnover, synthesis and breakdown were observed between the two groups, but protein accretion was 71% to 79% higher in the preterm formula group than the fortified human milk group. Urea production rates were not different in the two groups. There was a strong negative correlation between urea production and protein accretion calculated from phenylalanine kinetics but not when calculated from valine kinetics. Preterm formula and fortified human milk appear equally well tolerated by preterm small for gestational age neonates, but protein accretion was higher in the preterm formula group. In preterm small for gestational age infants, both phenylalanine and valine kinetic methods can be used to accurately determine protein metabolism.

Altered Turnover of Calcium Regulatory Proteins of the Sarcoplasmic Reticulum in Aged Skeletal Muscle

We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 +/- 0.4, 6.3 +/- 1.3, and 8.3 +/- 1.3 days, respectively. A half-life of 14.5 +/- 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 +/- 5% and 25 +/- 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 +/- 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.

Effects of heat stress on enzyme activities and transcript levels in developing maize kernels grown in culture

Heat stress can cause decreased grain yields and increased susceptibility in maize to the aflatoxin-producing fungus, Aspergillus flavus. We hypothesized that heat stress may affect gene expression in developing kernels, thereby affecting enzyme activities and storage product accumulation. To test this hypothesis, maize ( Zea mays L. hybrid B73xMo17) kernels were cultured in vitro and grown at 25 (control), 30, and 35°C. At 5-day intervals, developing kernels were analyzed for levels of gene expression and enzyme activity. Message levels of all genes evaluated, including Shrunken-1, Shrunken-2, Brittle-2, Aldolase, Waxy, α-Zein, β-Zein, and Opaque-2 were reduced 65 to 80% after 5 days at 35°C. In contrast, of the 17 enzymes assayed, only the activities of adenosine diphosphate glucose (ADP-Glc) pyrophosphorylase, aldolase, aspartate aminotransferase, acid invertase, and acid phosphatase were decreased by heat-stress. Although the decrease in ADP-Glc pyrophosphorylase activity corresponded well with its decreased mRNA, aldolase activity only loosely corresponded to its mRNA levels, and sucrose synthase activity was not affected by growth temperature, although its transcript was reduced by 80%. We suggest that the declines in enzyme activities in the heat-stressed kernels are a result of declines in transcript. The differing responses of enzyme activities to the heat stress may reflect differences in protein turnover. In a subsequent experiment, we tested the hypothesis that heat stress may interfere with the ability of a maize kernel to generate pathogenesis-response proteins. We found that the activity of the pathogenesis-related protein, chitinase, was increased two-fold in cultured kernels grown at 25°C by treatment with salicylic acid. However, the salicylate-induced increase in chitinase activity was inhibited in kernels grown at 35°C. These results suggest that heat stress inhibits the induction of pathogenesis-related proteins in the maize kernel, and may, thereby, interfere with the ability of the kernel to defeat pathogen invasion.

Glutamine supplementation of enteral nutrition: impact on whole body protein kinetics and glucose metabolism in critically ill patients.

Glutamine-supplemented parenteral nutrition has been reported to attenuate the early postoperative reduction in intracellular glutamine and improve protein synthesis and nitrogen balance. We investigated the effect of an enteral formula or protein and glucose kinetics and nitrogen balance in trauma patients. The enteral formula (AlitraQ) provided a mean intake of 0.35 g of glutamine/kg body weight per day to 16 trauma patients and was compared with an isonitrogenous formula that provided a mean of 0.05 g of glutamine/kg body weight per day in 14 trauma patients. After 3 days of feeding, protein kinetics were measured using a 4-hour prime-continuous infusion of L-[1-13C]leucine. Glucose kinetics were measured during the same time interval using prime-continuous infusion of [U-14C]- and [6-3H]glucose. Nitrogen balance was not significantly different in the two groups. There were no significant differences in protein turnover, synthesis, and breakdown between the two groups. There were no significant differences in glucose turnover, oxidation, recycling, and percent of VCO2 from glucose oxidation between the two groups. Glutamine-enriched enteral formulas are well tolerated by the severely injured patient but provide no additional nutritional advantage compared with standard enteral formulas during the first 3 days of feeding immediately after trauma.

Individual variation in protein turnover and growth efficiency in rainbow trout, Oncorhynchus mykiss (Walbaum)

The aims of this study were to examine the relation between consumption, growth and protein synthesis for individual rainbow trout held in groups, and to examine how differences in protein turnover may help to explain the observed differences in protein growth rate between rainbow trout with similar protein consumption rates. Therefore four measurements of consumption were made for 37 individual fish over a 73 day period by using radiography. At the end of the experiment, fractional rates of protein synthesis were measured by using a single flooding dose injection of [$^{3}$H]phenylalanine. The results showed that fractional rates of protein growth and synthesis increased with consumption rate. However, degradation rates were independent of consumption. Fish consuming similar amounts of food exhibited two- to threefold differences in fractional protein growth rates, and twofold differences in fractional rates of protein synthesis. Nine pairs of fish with similar protein consumption rates but different rates of protein growth were selected to examine if differences in protein metabolism, digestive efficiency or RNA concentration or activity could explain these observed differences in protein growth efficiency. More efficient fish were found to have reduced degradation rates; this was the only significant result obtained. The results support the hypothesis that individual differences in protein turnover are important determinants of growth efficiency in fish.

Coordination of expression of Zymomonas mobilis glycolytic and fermentative enzymes: a simple hypothesis based on mRNA stability.

Although Zymomonas mobilis is prototrophic, glycolytic and fermentative enzymes (ethanologenic enzymes) constitute over half of the cytoplasmic protein. In this study, transcript stability, functional message pools, and the abundance of cytoplasmic products were compared for genes encoding eight of these essential enzymes. The transcripts of all were very stable, with half-lives ranging from 8 to 18 min. This transcript stability is proposed as an important feature in Z. mobilis that may distinguish highly expressed genes for energy generation from biosynthetic genes, which are required at much lower levels. The evolution of multiple promoters to enhance transcription from single-copy genes, of structural features that alter translational efficiency, and of differences in protein turnover is hypothesized to serve a subordinate role in the regulation of Z. mobilis gene expression. Among the eight ethanologenic genes examined, differences in transcript stability were found to directly correlate with differences in functional message pools and cytoplasmic protein levels. These differences in transcript stability are hypothesized to have evolved as a primary mechanism to balance the levels of individual enzymes within the glycolytic and fermentative pathways.

Effect of chronic ethanol administration on protein catabolism in rat liver.

Hepatic protein catabolism was measured in rats which were pair-fed a liquid diet containing either ethanol or isocaloric maltose-dextrin (control diet). Within 12 days after initiation of pair feeding, the level of total hepatic protein in ethanol-fed rats was 26% higher than that in pair-fed control rats. During this time interval, the catabolic rates of both short-lived [3H]puromycin-labeled proteins and long-lived native [14C]bicarbonate-labeled proteins were measured in the two groups of animals. The degradation rate of short-lived [3H]puromycinyl proteins and peptides was the same in ethanol-fed and pair-fed control rats. However, the overall catabolic rate of long-lived proteins in rats fed the ethanol liquid diet for 2-10 days was 37-40% lower than that in pair-fed controls. This difference in protein turnover was not a general phenomenon, since the time-dependent decay of [14C]proteins in the hepatic microsome fraction of ethanol-fed rats was 33% slower than that in pair-fed controls, but the apparent rate of cytosolic protein catabolism was the same in both groups of animals. The differences in protein turnover did not reflect quantitative changes in lysosomal proteases since the activities of four hepatic lysosomal cathepsins were unaffected by alcohol administration. When rats were subjected to longer periods of pair feeding (16-25 days), the difference in overall hepatic protein catabolism between ethanol-fed rats and their pair-fed controls was considerably attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-lapse cinematographic studies on the inhibition of cell motility by puromycin in cultured nervous tissue

Samples of mouse cerebellar cortex were cultivated in Maximow slides and Rose chambers with TC medium 1066 (Difco) and investigated by cinematographic techniques. The blockade of protein synthesis by puromycin produces different effects on cell motility depending on the cellular type. The mesenchymal cells show, for example, a reversible inhibition of their locomotion and their ruffling membrane activity under the influence of a given concentration of puromycin. The same dose of puromycin inhibits the pulsating activity of glial cells gradually, followed by total paralysis. This effect on glial cells is not reversible. The effects of the same puromycin dose on neurons are hardly recognizable with the technique used. The different responses by glia cells, neurons and mesenchymal cells to protein synthesis inhibition by puromycin may possibly be due to differences in protein turnover by these cells. These results suggest that there may exist a direct relation between protein synthesis and cell motility.