Fractional Synthesis Rate Research Articles

The Anabolic Response to Dietary Protein Is Not Limited by the Maximal Stimulation of Protein Synthesis in Healthy Older Adults: A Randomized Crossover Trial

We have recently demonstrated in young adults that an anabolic response with mixed meal protein intake above ~35 g/meal, previously recognized as an “optimal” protein dose, was further stimulated. However, it is unknown if this applies to older adults. We therefore examined anabolic response to a mixed meal containing either 35 g (MOD, moderate amount of protein) or 70 g (HIGH, high amount of protein) in a randomized cross-over metabolic study in older adults (n = 8). Primed continuous infusions of L-[2H5] phenylalanine and L-[2H2]tyrosine were performed to determine whole-body protein kinetics and muscle protein fractional synthesis rate (MPS) in basal fasted and fed states. Whole-body protein kinetics (NB, net protein balance; PS, protein synthesis; PB, protein breakdown) and MPS was expressed as changes from the baseline post-absorptive state. Consistent with our previous findings in young adults, both feedings resulted in a positive NB, with HIGH being more positive than MOD. Furthermore, NB (expressed as g protein∙240 min) increased linearly with an increasing amount of protein intake, expressed relative to lean body mass. The positive NB was achieved due mainly to the suppression of PB in both MOD and to a greater extent HIGH, while PS was only increased in HIGH. Consistent with the whole-body data, MPS was significantly higher in HIGH than MOD. Plasma concentrations of essential amino acids and insulin were greater in HIGH vs. MOD. We conclude that in the context of mixed meals, whole-body anabolic response linearly increases with increasing protein intake primarily through the suppression of PB, and MPS was further stimulated with protein intake above the previously considered “optimal” protein dose in older adults.

Mitochondrial Function and Protein Turnover in the Diaphragm are Altered in LLC Tumor Model of Cancer Cachexia.

It is established that cancer cachexia causes limb muscle atrophy and is strongly associated with morbidity and mortality; less is known about how the development of cachexia impacts the diaphragm. The purpose of this study was to investigate cellular signaling mechanisms related to mitochondrial function, reactive oxygen species (ROS) production, and protein synthesis during the development of cancer cachexia. C57BL/J6 mice developed Lewis Lung Carcinoma for either 0 weeks (Control), 1 week, 2 weeks, 3 weeks, or 4 weeks. At designated time points, diaphragms were harvested and analyzed. Mitochondrial respiratory control ratio was ~50% lower in experimental groups, which was significant by 2 weeks of cancer development, with no difference in mitochondrial content markers COXIV or VDAC. Compared to the controls, ROS was 4-fold elevated in 2-week animals but then was not different at later time points. Only one antioxidant protein, GPX3, was altered by cancer development (~70% lower in experimental groups). Protein synthesis, measured by a fractional synthesis rate, appeared to become progressively lower with the cancer duration, but the mean difference was not significant. The development and progression of cancer cachexia induces marked alterations to mitochondrial function and ROS production in the diaphragm and may contribute to increased cachexia-associated morbidity and mortality.

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.

In vivo collagen and mixed muscle protein turnover in 2 meat-type broiler strains in relation to woody breast myopathy

Two meat-type broiler strains, strain A and strain B, were reared in floor pens (25 birds/pen; 45 pens/strain) for pectoralis (P) major collagen and mixed muscle protein turnover (PT) study from 0–56 D using primary breeder nutrition and husbandry guidelines. Forty broilers (n = 10/strain for collagen PT; n = 10/strain for mixed muscle PT) were selected at each sampling age at day 21, 28, 35, 42, and 56 and infused with 1-13C proline (Pro) and 15N-phenylalanine (Phe) which are used as amino acid tracers for collagen and mixed muscle PT measurements, respectively. Muscle and plasma samples were collected, and enrichments of 1-13C Pro and 15N-Phe were determined using mass spectrometry. Fractional synthesis rate (FSR) and fractional degradation rate (FDR) were measured for collagen and mixed muscle using precursor-product principle. At day 42, after separating the sampled broilers as myopathy (woody breast [WB] score > 1) and nonmyopathy (WB = 0), plasma metabolites were screened for differential 3-methyhistidine (3-MH) expression for both strains. Data were analyzed using one-way ANOVA using t test. Results showed that collagen and mixed muscle FSR and FDR in pectoralis major decreased (P < 0.05) for both strains as the broilers aged. FSR for collagen and FDR for mixed muscle were higher for strain B than those for strain A (P < 0.05). Total collagen was higher (P < 0.05) for strain B. Differentially expressed 3-MH in plasma was higher (P < 0.05) for myopathy-affected broilers indicating greater muscle degradation occurring in myopathy-affected broiler types for both strains. 3-MH Expression in plasma was higher for strain B than for strain A. The research findings showing an increased collagen content per unit muscle weight in pectoralis major in strain B (than in strain A) could be due to higher mixed muscle FDR and increased collagen FSR occurring during the grow-out period. The increased degradation of muscle fibers and probable replacement of muscle-specific protein with connective tissue, mainly collagen, was an evident pathophysiological phenomenon occurring in myopathy-affected broilers.

A Ketogenic Diet In Mice Reduces Cardiac Protein Synthesis Compared to a Western Diet

Abstract Objectives For several decades, the ketogenic diet has been studied as a replacement for the western diet for weight management and disease treatment. However, there is a lack of understanding regarding the mechanism of change in metabolism induced by the diet. To better understand the diet's physiological effects, we hypothesize that protein turnover in specific organs, like the heart, are modified by the different diets. Therefore, we studied organ protein fractional synthesis rate (FSR) in several organ tissues of mice given ketogenic and a control western diets. Methods To assess protein FSR of various organs, we studied 4–6 week-old A/J mice) and randomly provided a ketogenic diet (high fat, no carbohydrate, n = 10) or a western diet (high fat, high carbohydrate, n = 10) for 3 months. One day before tissue collection, a D2O bolus was administered via intraperitoneal injection, and mice were provided D2O enriched drinking water to enrich the total body water to about 5% D2O. Eleven tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected and analyzed for alanine enrichment in the intracellular and protein-bound pool (LC-MS/MS). FSR was calculated as -ln(1-enrichment) as fraction per day. Data are expressed as mean ± SE (unpaired t-test: GraphPad Prism 8.2). Results We found a significantly lower heart protein FSR in ketogenic diet compared to the western diet (ketogenic: 0.0795 ± 0.0021, western: 0.0873 ± 0.0015, P = 0.0076). We did not find any difference between protein FSR of the ketogenic and western diet mice in any of the other measured organs. Conclusions The ketogenic diet in mice reduces cardiac protein synthesis, which could potentially indicate benefits of a ketogenic diet. We hypothesize that the mechanism of a ketogenic diet to reduce cardiac protein synthesis is via reduced inflammation and normalized heart function (e.g., reduced blood pressure). Funding Sources Sydney and J.L. Huffines Institute for Sports Medicine, Human Performance Student Research Grant and CTRAL Grant.

Comparable Organ Protein Fractional Synthesis Rate of High and Low-Active Mice

Abstract Objectives With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Scientists have explored physical activity regulation by investigating various physiological mechanisms involving hormones, neurotransmitters, and genetics; however, little is known about the role of metabolism on physical activity level. We hypothesize that protein turnover in specific organs like the muscle is higher in mice previously exhibiting high physical activity levels, as a mechanism to adapt to the increased demand. Therefore, we studied protein fractional synthesis rate (FSR) in tissues of inherently high and low active mice. Methods In order to study protein FSR of various organs, we assessed 12-week-old male inherently low-active (LA) mice (n = 23, lean body mass: 21.0 ± 1.1 g, C3H/HeJ strain) and high active (HA) mice (n = 20, lean body mass: 22.5 ± 1.3, C57L/J strain). One day before tissue collection, a D2O bolus was administered via intraperitoneal injection, and mice were provided D2O enriched drinking water to enrich the total body water to about 5% D2O. Eleven tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected and analyzed for enrichment of alanine in the intracellular and protein-bound pool (LC-MS/MS). FSR was calculated as -ln(1-enrichment) as fraction per day. Data are mean ± SE (unpaired t-test: GraphPad Prism 8.2). Results We did not find significant differences between protein FSR of HA and LA mice in any measured organ. Example: Protein FSR (fraction/day): muscle (LA: 0.0326±-0.0026, HA: 0.0331 ± 0.0018, P = 0.8673), liver (0.3568 ± 0.0219, 0.3499 ± 0.0217, P = 0.8263), brain (0.0981 ± 0.0056, 0.1041 ± 0.0063, P = 0.4758). Conclusions The observed lack of significant differences in high and low-active mice suggests that differences in specific organ tissue protein turnover may not be a mechanism regulating inherent physical activity level. Since protein turnover is representative of the ability to adapt through upregulation and downregulation of metabolic processes, these results show that high-active mice are inherently no more equipped for metabolic regulation than the low active mice. Funding Sources Sydney and J.L. Huffines Institute for Sports Medicine, Human Performance Student Research Grant and CTRAL Grant.

Effects of Testosterone on Mixed Muscle Protein Synthesis and Proteome Dynamics During Energy Deficit

Abstract Objectives Short-term energy deficit reduces acute measures of mixed muscle protein synthesis (MPS) and suppresses the hypothalamic-pituitary axis and endogenous testosterone synthesis. We hypothesized that testosterone supplementation could mitigate the effects of energy deficit on MPS. We conducted a randomized, double-blind, placebo-controlled trial to determine the effects of 28 days of tightly-controlled severe energy deficit (deficit 55% of total energy requirements) on measures of mixed-MPS and proteome-wide protein dynamics in non-obese men either given 200 mg testosterone enanthate (Testosterone, n = 24) or placebo (Placebo, n = 26) injections per week. Methods Participants received daily aliquots of deuterated water (2H2O) for 42 consecutive days (14-d weight maintenance period followed by 28-d energy deficit). Muscle biopsies were collected at rest in a fasted state at the end of the weight maintenance phase (PRE) and at the middle (MID) and end (POST) of the 28-d energy deficit. Mixed-MPS and proteome-wide protein fractional synthesis rates (FSR) were quantified. Changes over time and differences between Testosterone and Placebo were determined for mixed-MPS, and cross-sectional comparisons between Testosterone and Placebo were performed at MID and POST for proteome dynamics. Results In both Testosterone and Placebo, mixed-MPS were 40% and 33% lower (P &amp;lt; 0.0005) at MID and POST energy deficit, respectively, compared to PRE, with no differences between groups or between MID and POST. Proteome-wide FSR of individual muscle proteins did not differ between Testosterone and Placebo at any time point. However, at POST, the number of individual proteins with higher FSR in Testosterone than Placebo was significant by 2-tailed binomial test (P &amp;lt; 0.05), with values ranging from 20–32% higher FSR for myofibrillar, mitochondrial and cytosolic proteins. Conclusions Findings confirm the pronounced effect of short-term severe energy deficit on mixed-MPS and suggest the anabolic suppression occurs largely independent of testosterone. However, proteome-wide protein dynamics may reveal a novel time sensitive signal by which supplemental testosterone triggers a delayed increase in MPS, providing a synthetic mechanism for muscle mass preservation or accrual. Funding Sources Supported by DHP JPC-5/MOMRP; authors’ views not official U.S. Army or DoD policy.

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles.

Differences in the protein composition of fast- and slow-twitch muscle may be maintained by different rates of protein turnover. We investigated protein turnover rates in slow-twitch soleus and fast-twitch plantaris of male Wistar rats (body weight 412 ± 69 g). Animals were assigned to four groups (n = 3, in each), including a control group (0 d) and three groups that received deuterium oxide (D2O) for either 10 days, 20 days or 30 days. D2O administration was initiated by an intraperitoneal injection of 20 μL of 99% D2O-saline per g body weight, and maintained by provision of 4% (v/v) D2O in the drinking water available ad libitum. Soluble proteins from harvested muscles were analysed by liquid chromatography–tandem mass spectrometry and identified against the SwissProt database. The enrichment of D2O and rate constant (k) of protein synthesis was calculated from the abundance of peptide mass isotopomers. The fractional synthesis rate (FSR) of 44 proteins in soleus and 34 proteins in plantaris spanned from 0.58%/day (CO1A1: Collagen alpha-1 chain) to 5.40%/day NDRG2 (N-myc downstream-regulated gene 2 protein). Eight out of 18 proteins identified in both muscles had a different FSR in soleus than in plantaris (p < 0.05).

SAT-LB95 Low Density Lipoprotein Receptor and Proprotein Convertase Subtilisin/Kexin Type 9 Kinetics Using Heavy Water (2H2O) Labeling and Mass Spectrometry

Abnormally high blood cholesterol levels in low density lipoprotein (LDL) increases the risk of heart disease. Cell surface receptors such as LDL-receptors (LDLr) regulate the clearance of LDL from blood circulation. As cholesterol levels decrease, cells promote cholesterol synthesis and cholesterol uptake by increasing LDLr expression. Another regulatory protein of plasma cholesterol clearance is proprotein convertase subtilisin/kexin type 9 (PCSK9). It is secreted from the liver into circulation where it can bind to and target LDLr to the lysosome for subsequent degradation. The current model of cholesterol regulation describes how increased cholesterol content down-regulates the number of LDLr promoted by PCSK9 mediated degradation, however minimal knowledge is not known about LDLr and PCSK9 kinetics using heavy water labeling, and how cholesterol enriched diet affects LDLr and PCSK9 kinetics in vivo. Therefore, our objective(s) were to establish a method 1) to measure the kinetics of LDLr and PCSK9 via stable isotopic metabolic labeling with heavy water (2H2O) in vivo 2) to further test established models of cholesterol metabolic regulation on LDLr and PCSK9 turnover after feeding mice a cholesterol enriched diet. We hypothesize that a cholesterol enriched diet will decrease both LDLr and PCSK9 synthesis rates. In order to test this, mice were fed a cholesterol enrich diet for 1 week and metabolically labeled with heavy water (2H2O) up to 36 hours. LDLr and PCSK9 were immunoprecipitated from liver and deuterium incorporation into LDLr and PCSK9 were measured via mass spectrometry. Our results revealed high cholesterol feeding down-regulated cholesterol synthesis and LDLr fractional synthesis rate decreased from 10.0% to 6% per hour. PCSK9 concentration also decreased from 1 to 0.2 (ng/ml / total mg protein), but the synthesis rate increased from 9.0%/day in control mice to 19.5%/day in high cholesterol diet. These results suggest high cholesterol feeding increases PCSK9 synthesis that potentially depletes the intracellular pool to target LDLr to the lysosome thus decreasing LDLr turnover. This research provides a flux-based approach to measure the kinetics of LDLr and PCSK9 for a molecular based kinetic insight of their functions in physiology, disease and therapy.

Possibilities of modern drug correction of metabolic syndrome: the role of bile acids

After the discovery of the method of synthesis of ursodeoxycholic acid (UDCA) and the publication of evidence confirming its ability to reduce the lithogenic properties of bile, active clinical use of UDCA began around the world. This drug, in addition to the proven choleretic, cytoprotective, litholytic, anti-apoptotic effects, has a signaling activity that allows UDCA to influence metabolic syndrome components such as hyperglycemia, hypercholesterolemia.&#x0D; Under the influence of UDCA, FXR is activated in the liver, which leads to an increase in the activity of glycogen synthase and decrease in the level of glycaemia. Another mechanism by which UDCA affects glycaemia is mediated by the activation of the TGR5 membrane receptor under the influence of this bile acid, as well as the release of insulin from pancreatic β-cells and decrease in postprandial glycaemia. When taking UDCA, the concentration of glycosylated hemoglobin, insulin in the blood plasma decreases the effects of insulin resistance decrease. UDCA has a beneficial effect on the vascular wall, reducing the severity of atherosclerotic lesions and normalizing the average thickness of the intima-media complex. UDCA improves lipid metabolism by regulating the activity of the AKT/ mTOR-signaling pathway, reduces the synthesis of cholesterol, and decreases the fractional rate of cholesterol synthesis and the fractional rate of triglyceride synthesis. It is proved that UDCA administration is accompanied by a drop in the level of total cholesterol and cholesterol of low-density lipoproteins. Normalization of the metabolism of glucose, triglycerides, cholesterol and the insulin-signaling pathway under the influence of bile acids is the basis for the use of UDCA for the correction of metabolic syndrome, as well as its hepatological manifestations - NAFLD.

Dynamic Proteome Profiling of Protein Fractional and Molar Synthesis Rates in Human Muscle in vivo

This study aimed to develop a method for measuring the abundance and synthesis rate of human muscle proteins in molar rather than fractional (%/d) terms. Losses in proteostasis are implicated in ageing and chronic diseases but research is hindered by a lack of methods for studying protein dynamics in humans. Protein‐specific fractional synthesis rate (FSR) can be measured by stable isotope labelling and peptide mass spectrometry (MS). However, FSR is a measurement of an unknown whole, i.e. protein abundance. If protein abundance changes or is different between conditions, the interpretation of FSR data may be confounded.Three physically active males (21 ± 1 years; height 178 ± 1 cm; weight 75 ± 5 kg) gave their informed consent to the ethically approved procedures. Newly synthesised proteins were labelled by oral consumption of deuterium oxide (50 ml of 99.8 atom %) taken four times per day during days 1–5, and two times per day during days 6–12. Samples of blood and vastus lateralis were collected after an overnight fast on days 0, 4, 8 and 12. Precursor enrichment and incorporation of deuterium oxide into muscle proteins were determined by liquid chromatography tandem MS. Protein abundances were normalised to 30 fmol yeast alcohol dehydrogenase added during sample preparation. FSR (%/d) was calculated from time‐dependent changes in peptide mass isotopomer abundances. FSR and abundance data (fmol/ μg protein) were combined to calculate molar synthesis rates (MSR; fmol/ μg protein/ d).Abundance data were collected for 1,368 proteins that had at least 1 unique peptide. FSR data were calculated from 3,944 peptides representing 940 proteins (average 3 peptides per protein, range 1 – 53 peptides per protein). In total, 769 proteins had abundance and FSR data in two or more participants. The median (M), lower‐(Q1) and upper‐quartile (Q3) values for protein FSR (%/d) were M = 4.39, Q1 = 0.96, Q3 = 7.84. Alpha skeletal muscle actin had the lowest FSR (0.28 ± 0.1 %/d) whereas the highest recorded FSR was 54.43 ± 8.9 %/d (nuclear protein localization protein 4 homolog). tRNA ligases were common amongst top‐ranked proteins by FSR but were ranked amongst the lowest in abundance e.g. Leucine‐‐tRNA ligase was the lowest abundance (5 amol/ μg protein) protein detected. The most abundant protein was myoglobin (1.52 pmol/ μg protein). Average MSR (fmol/ μg protein/ d) was M = 0.05 (Q1 = 0.017, Q3 = 0.13). Metabolic enzymes were amongst the top‐ranked proteins by MSR (range 1–10 fmol/ μg protein/ d) alongside cullin‐associated NEDD8‐dissociated protein 2 (0.9 ± 0.2 fmol/ μg protein/ d) and antioxidant enzymes, including mitochondrial superoxide dismutase [Mn], peroxiredoxin 3, peroxiredoxin 2 and glutathione S‐transferase‐P (each ~0.5 fmol/ μg protein/ d).MSR proteomic data offers new insight to the allocation of cellular resources. Future applications investigating muscle adaptation or differences between health and disease may uncover unexpected insight regarding which proteins are most synthesised and highlight new avenues of research in human muscle physiology.

Effect of ursodeoxycholic acid on lipid metabolism: through the prism of evidence from 2019.

After the discovery of the method of ursodeoxycholic acid’s (UDCA) synthesis and the publication of evidence confirming its ability to reduce the lithogenic properties of bile, active clinical use of UDCA began in the world. This drug, which has pleiotropic effect (choleretic, cytoprotective, immunomodulatory, antiapoptic, litholytic, hypocholesterolemic), has proven its effectiveness in the treatment various diseases: primary biliary cholangitis, intrahepatic cholestasis of pregnancy, gallstone disease.&#x0D; Being a tertiary bile acid, UDCA stimulates bile acid synthesis by reducing the circulating fibroblast growth factor 19 and inhibiting the activation of the farnesoid X-receptor (FXR), which leads to the induction of cholesterol-7α-hydroxylase, a key enzyme in the synthesis of bile acid de novo, mediating the conversion of cholesterol into bile acids. Changes in the formation of bile acids and cholesterol while taking UDCA intake is accompanied by activation of the main enzyme of cholesterol synthesis - 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Under the influence of UDCA the activity of stearoyl-Coa desaturase (SCD) in visceral white adipose tissue increases. According to studies conducted in 2019, UDCA improves lipid metabolism by regulating the activity of the ACT/mTOR signaling pathway, reduces the synthesis of cholesterol, decreases the fractional synthesis rate of cholesterol and the fractional synthesis rate of triglycerides. It has been proved that UDCA is accompanied by a decrease in the level of total cholesterol and low density lipoprotein cholesterol.

Polyvinylpyrolidone-functionalized silver nanoparticles do not affect aerobic performance or fractional rates of protein synthesis in rainbow trout (Oncorhynchus mykiss)

Aerobic performance in fish is linked to individual and population fitness and can be impacted by anthropogenic contaminants. Exposure to some engineered nanomaterials, including silver nanoparticles (nAg), reduces rates of oxygen consumption in some fish species, but the underlying mechanisms remain unclear. In addition, their effects on swim performance have not been studied. Our aim was to quantify the impact of exposure to functionalized nAg on aerobic scope and swim performance in rainbow trout (Oncorhychus mykiss) and to characterize the contribution of changing rates of protein synthesis to these physiological endpoints. Fish were exposed for 48 h to 5 nm polyvinylpyrolidone-functionalized nAg (nAgPVP; 100 μg L−1) or 0.22 μg L−1 Ag+ (as AgNO3), which was the measured quantity of Ag released from the nAgPVP over that time period. Aerobic scope, critical swimming speed (Ucrit), and fractional rates of protein synthesis (Ks), were then assessed, along with indicators of osmoregulation and cardiotoxicity. Neither nAgPVP, nor Ag+ exposure significantly altered aerobic scope, its component parts, or swim performance. Ks was similarly unaffected in 8 tissue types, though it tended to be lower in liver of nAgPVP treated fish. The treatments tended to decrease gill Na+/K+-ATPase activity, but effects were not significant. The latter results suggest that a longer or more concentrated nAgPVP exposure may induce significant effects. Although this same formulation of nAgPVP is bioactive in other fish, it had no effects on rainbow trout under the conditions tested. Such findings on common model animals like trout may thus misrepresent the safety of nAg to more sensitive species.

In-vivo Intramuscular Collagen Synthesis, Muscle Fiber Growth and Histomorphology of Pectoralis major of a Fast-Growing Broiler Strain Gallus gallus domesticus.

Collagen protein has been considered as major culprit to myopathy condition affecting Pectoralis major, called woody breast (WB) in Gallus gallus domesticus (broiler). The WB myopathy is characterized by macroscopic stiffness of P. major and the affected tissue have reduced protein quality. This study measured the in-vivo soluble (S-) and insoluble (I-) collagen fractional synthesis and degradation rates (FSR and FDR) in P. major over typical grow-out cycle of broiler using stable isotope of 1-13C proline as metabolic tracer. Collagen content and muscle fiber histomorphology of P. major were also assessed simultaneously. The FSR and FDR for S- and I-collagen decreased over age, however FSR remained higher than FDR suggesting collagen was accreting during the grow-out period. This was reflected by increment in total collagen content in P. major in maturing broiler. Histomicrographs showed myodegeneration occurring as early as 21 days followed by greater accumulation of collagenous tissue in perimysial and endomysial connective tissue spaces of muscle fibers as bird aged. The findings suggest that reduced turnover of collagen in P. major at the later age of bird could have evolved due to adaptive physiological feedback mechanism against further synthesis and deposition of collagen in the extracellular matrix.

The addition of an amylopectin/chromium complex to branched-chain amino acids enhances muscle protein synthesis in rat skeletal muscle

BackgroundA previous clinical study reported that the addition of an amylopectin/chromium complex (ACr; Velositol®) to 6 g of whey protein (WP) significantly enhanced muscle protein synthesis (MPS). Branched-chain amino acids (BCAAs) are also well-known to enhance MPS. The aim of this study was to determine if the addition of ACr to BCAAs can enhance MPS and activate expression of the mammalian target of the rapamycin (mTOR) pathway compared to BCAAs and exercise alone in exercise-trained rats.MethodsTwenty-four male Wistar rats were randomly divided into three groups (n = 8 per group): (I) Exercise control, (II) Exercise plus BCAAs (0.465 g/kg BW, a 6 g human equivalent dose (HED)), and (III) Exercise plus BCAAs (0.465 g/kg BW) and ACr (0.155 g/kg BW, a 2 g HED). All animals were trained with treadmill exercise for 10 days. On the day of the single-dose experiment, rats were exercised at 26 m/min for 2 h and then fed, via oral gavage, study product. One hour after the consumption of study product, rats were injected with a bolus dose (250 mg/kg BW, 25 g/L) of phenylalanine labeled with deuterium to measure the fractional rate of protein synthesis (FSR). Ten minutes later, muscle tissue samples were taken to determine MPS measured by FSR and the phosphorylation of proteins involved in the mTOR pathway including mTOR, S6K1, and 4E-BP1.ResultsACr combined with BCAAs increased MPS by 71% compared to the exercise control group, while BCAAs alone increased MPS by 57% over control (p < 0.05). ACr plus BCAAs significantly enhanced phosphorylation of mTOR, S6K1 and 4E-BP1 compared to exercise control rats (p < 0.05). The addition of ACr to BCAAs enhanced insulin levels, mTOR and S6K1 phosphorylation compared to BCAAs alone (p < 0.05). Serum insulin concentration was positively correlated with the levels of mTOR, (r = 0.923), S6K1 (r = 0.814) and 4E-BP1 (r = 0.953).ConclusionsIn conclusion, the results of this study provide evidence that the addition of ACr to BCAAs significantly enhances exercise-induced MPS, and the phosphorylation of mTOR signaling proteins, compared to BCAAs and exercise alone.

In vivo kinetic study of the materno-fetal fatty acid transfer in obese and normal weight pregnant women

AbstractIntroduction:Placental function can be modified by maternal obesity affecting the materno-fetal fatty acid transport process. We analyze for the first time the in vivo materno-fetal kinetic transfer of fatty acids (FA) labeled with stable isotopes in control and obese pregnant women.Materials and Methods:Labelled FA with similar metabolism (stearic acid: 13C-SA, palmitic acid: 13C-PA and oleic acid: 13C-OA) were orally administered at -4 h, -8 h, and -12 h, respectively prior to elective caesarean section to 10 pregnant women with a body mass index (BMI) &gt; 30 and 10 with BMI 25–30 (control). Placenta, venous and arterial cord blood were collected obtaining a wide range of FA enrichments. A combined experimental and computational modeling analysis was applied.Results:FA Fractional synthesis rate (FSR) in placenta was 11–12% per hour. No differences were observed between control and normo-lipidemic obese pregnant women. It was not possible to estimate FA FSR in cord blood with this oral bolus dose approach. Computational modelling demonstrated a good fit to the data when all maternal plasma lipid classes were included but not with modelling based only on the non-esterified FA fraction (NEFA). The estimated materno-fetal 13C-FA transfer was around 1%.Conclusions:Our approach using multiple 13C-FA tracers allowed us to estimated FSR in placental/maternal plasma but not in fetal/maternal compartments. Computational modelling showed a consistent time course of placental 13C-FA transfer and predicted total fetal FA accumulation during the experiment. We conclude that in addition to NEFA in the maternal circulation, maternal plasma VLDL and other lipoproteins are important contributors to placental FA transfer to the fetus.

Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC-MS analysis is performed on muscle samples taken from male participants (n=4) that consumed 4×50mL doses of deuterium oxide (2 H2 O) per day for 14 days. ABD is measured by label-free quantitation and FSR is calculated from time-dependent changes in peptide mass isotopomer abundances. One-hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty-four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower-, (Q1 ) and upper-quartile (Q3 ) values for protein FSR (%/d) are M=1.63, Q1 =1.07, and Q3 =3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q1 1.7% to Q3 6.7%), whereas the median CV of FSR data is 10.1% (Q1 3.5% to Q3 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.

The isotopic nitrogen turnover rate as a proxy to evaluate in the long-term the protein turnover in growing ruminants

AbstractProtein turnover is an energy-consuming process that is essential for ensuring the maintenance of living organisms. Gold standard methods for whole-body protein turnover (WBPT) measurement have inherent drawbacks precluding their generalization for large farm animals and use during long periods. Here, we proposed a non-invasive proxy for the WBPT over a long period of time and in a large number of beef cattle. The proxy is based on the rate at which urine-N and plasma proteins are progressively depleted in terms of15N after a slight decrease in the isotopic N composition of the diet (i.e. diet switch). We aimed to test the ability of this proxy to adequately discriminate the WBPT of 36 growing-fattening young bulls assigned to different dietary treatments known to impact the WBPT rate, with different protein contents (normalv.high) and amino acid profiles (balancedv.unbalanced in methionine). The15N depletion rate found in plasma proteins represented their fractional synthesis rate, whereas the slow depletion rate found in urine was interpreted as a proxy of the WBPT. The proxy tested in urine suggested different WBPT values between the normal- and high-protein diets but not between the balanced and unbalanced methionine diets. In contrast, the proxy tested in plasma indicated that both dietary conditions affected the fractional synthesis rate of plasma proteins. We considered that the rate at which urine is progressively15N-depleted following an isotopic diet switch could be proposed as a non-invasive proxy of the WBPT rate in large farm animals.

Protein synthesis rates of muscle, tendon, ligament, cartilage, and bone tissue in vivo in humans.

Skeletal muscle plasticity is reflected by a dynamic balance between protein synthesis and breakdown, with basal muscle tissue protein synthesis rates ranging between 0.02 and 0.09%/h. Though it is evident that other musculoskeletal tissues should also express some level of plasticity, data on protein synthesis rates of most of these tissues in vivo in humans is limited. Six otherwise healthy patients (62±3 y), scheduled to undergo unilateral total knee arthroplasty, were subjected to primed continuous intravenous infusions with L-[ring-13C6]-Phenylalanine throughout the surgical procedure. Tissue samples obtained during surgery included muscle, tendon, cruciate ligaments, cartilage, bone, menisci, fat, and synovium. Tissue-specific fractional protein synthesis rates (%/h) were assessed by measuring the incorporation of L-[ring-13C6]-Phenylalanine in tissue protein and were compared with muscle tissue protein synthesis rates using a paired t test. Tendon, bone, cartilage, Hoffa’s fat pad, anterior and posterior cruciate ligament, and menisci tissue protein synthesis rates averaged 0.06±0.01, 0.03±0.01, 0.04±0.01, 0.11±0.03, 0.07±0.02, 0.04±0.01, and 0.04±0.01%/h, respectively, and did not significantly differ from skeletal muscle protein synthesis rates (0.04±0.01%/h; P>0.05). Synovium derived protein (0.13±0.03%/h) and intercondylar notch bone tissue protein synthesis rates (0.03±0.01%/h) were respectively higher and lower compared to skeletal muscle protein synthesis rates (P<0.05 and P<0.01, respectively). Basal protein synthesis rates in various musculoskeletal tissues are within the same range of skeletal muscle protein synthesis rates, with fractional muscle, tendon, bone, cartilage, ligament, menisci, fat, and synovium protein synthesis rates ranging between 0.02 and 0.13% per hour in vivo in humans.Clinical trial registration: NTR5147

Prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of neonatal piglets.

Postnatal growth of lean mass is commonly blunted in preterm infants and may contribute to short- and long-term morbidities. To determine whether preterm birth alters the protein anabolic response to feeding, piglets were delivered at term or preterm, and fractional protein synthesis rates (Ks) were measured at 3 days of age while fasted or after an enteral meal. Activation of signaling pathways that regulate protein synthesis and degradation were determined. Relative body weight gain was lower in preterm than in term. Gestational age at birth (GAB) did not alter fasting plasma glucose or insulin, but when fed, plasma insulin and glucose rose more slowly, and reached peak value later, in preterm than in term. Feeding increased Ks in longissimus dorsi (LD) and gastrocnemius muscles, heart, pancreas, and kidney in both GAB groups, but the response was blunted in preterm. In diaphragm, lung, jejunum, and brain, feeding increased Ks regardless of GAB. Liver Ks was greater in preterm than term and increased with feeding regardless of GAB. In all tissues, changes in 4EBP1, S6K1, and PKB phosphorylation paralleled changes in Ks. In LD, eIF4E·eIF4G complex formation, phosphorylation of TSC2, mTOR, and rpS6, and association of mammalian target of rapamycin (mTOR1) complex with RagA, RagC, and Rheb were increased by feeding and blunted by prematurity. There were no differences among groups in LD protein degradation markers. Our results demonstrate that preterm birth reduces weight gain and the protein synthetic response to feeding in muscle, pancreas, and kidney, and this is associated with blunted insulin- and/or amino acid-induced translation initiation signaling.