Muscle Protein Synthesis Rates Research Articles

Recent advances for measurement of protein synthesis rates, use of the ‘Virtual Biopsy’ approach, and measurement of muscle mass

Flux-rate measurements of protein synthesis and breakdown (turnover) in muscle represent an ideal class of mechanism-based biomarkers for conditions of altered muscle mass and function. We describe here new metabolic labeling techniques for flux-rate measurements in humans, focusing on skeletal muscle. Dynamics of the muscle proteome are accurately measured in humans by combining long-term heavy water labeling with tandem mass spectrometry. Broad proteomic flux signatures or kinetics of targeted proteins are measurable. After interventions, early fractional synthesis rates of skeletal muscle proteins predict later changes in muscle mass. The 'virtual biopsy' method for measuring tissue protein turnover rates from body fluids has been validated for skeletal muscle, from labeling of plasma creatine kinase-type M or carbonic anhydrase-3. Label in these proteins in plasma reflects label of cognate proteins in the tissue, and response in plasma predicts longer term outcomes. Skeletal muscle mass can also be measured noninvasively from a spot urine, based on dilution of labeled creatine. This method correlates well with whole body MRI assessment of muscle mass and predicts clinical outcomes in older men. Flux measurements are available and more interpretable functionally than static measurements for several reasons, which are discussed.

Evaluating a Second Generation Phytochemical Nrf2 Activator on Proteostasis and Cytoprotective Gene Expression in Vivo

Aging is associated with increases in oxidative stress. Redox imbalance occurs when production of reactive oxygen species (ROS) exceeds the capacity of antioxidant enzymes to eliminate ROS. Increased levels of intracellular ROS can compromise proteostasis by causing irreversible damage to proteins. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) mediates the cellular endogenous antioxidant defense system by regulating antioxidant enzymes that are cytoprotective against ROS. Nrf2 can be activated phytochemically through the supplement Protandim. Previous work from our lab has demonstrated that a phytochemical based Nrf2 activator improves proteostasis in skeletal muscle in vivo. Recently, we have begun to characterize a second generation Nrf2 activator (PB125) that has increased anti-inflammatory action in addition to anti-oxidant properties. Since inflammation can blunt protein synthetic responses, we speculated that PB125 might provide additional benefits on proteostatic processes. PURPOSE: The purpose of the present study was to examine in vivo the effects of three different doses (10, 100, and 300 ppm) of PB125 supplementation on Nrf2 activation and proteostasis. METHODS: 60 male CB6F1 mice aged 10–11 months were assigned to diets containing low, medium, or high doses of product PB125 in a 5 week feeding study. Mice were isotopically labeled with 8% deuterium oxide (D2O) to simultaneously measure protein and DNA fractional synthesis rates (FSR) in liver, heart, and skeletal muscle. Nrf2 activation was assessed through analysis of gene expression profiles via Affymetrix GeneChip microarray. RESULTS: Proteostatic mechanisms were increased in the liver mitochondrial fraction in the 10 ppm treatment group (18.9 vs 16.3 FSR%/day, p<0.05). However, there were no differences in proteostatic mechanisms in heart or skeletal muscle. At 100 ppm, there was up-regulation of Nrf2-dependent cytoprotective genes (Akr1d1, Gpx2, Gclm, Fthl17b, 3.82, 1.84, 1.42, 1.64-fold increase). CONCLUSION: From our data we were able to conclude that all three doses were safe, and that 100 ppm was effective at activating Nrf2. In addition, there was an indication of increased proteostatic processes in the liver, but not heart or skeletal muscle, perhaps due to the healthy status of the mice.

Partial or Complete Unloading of Skeletal Muscle Leads to Specific Alterations of Anabolic Signal Transduction

Consequences of disuse atrophy of skeletal muscle observed during spaceflight on astronaut health and performance are a focal point of space research. Decrements of both muscle mass and protein synthesis rates have been observed with exposure to varying muscle loading environments (1G > partial loading > 0G), and most of the reduced muscle mass can be attributed to diminished rates of synthesis. However, specific mechanisms behind unloading-dependent reductions of protein synthesis are not well defined. PURPOSE: To determine whether or not alterations of anabolic signal transduction was responsible for the changes previously observed in fractional synthesis rates with specific gravitational loading paradigms. METHODS: Female BALB/cByJ were normalized by bodyweight and assigned to normal cage ambulation (1G), partial weight bearing suspension titrated to approximately 33% bodyweight (G/3), partial weight bearing titrated to 16% bodyweight (G/6) and full unloading of hind limbs (0G) in specially designed cages. All mice were subjected to that loading environment for 21d prior to tissue harvest, and monitored daily. Immunoblotting of the gastrocnemius (n=23) was carried out to analyze alterations of anabolic signal transduction. Although numerous signaling intermediates were assessed, the focus of this abstract will be on ribosomal protein S6 kinase (p70-S6K). This important protein has served as a marker of protein synthesis signal transduction as well as the anabolic capacity in skeletal muscle. RESULTS: Regardless of loading paradigm, no differences were detected among groups for the activation of p70-S6K (as indicated by the phospho: total protein content). Total protein content, however, was ~27% lower than control in 0G and G/6 (P=0.008) with G/3 not being different from control (P>0.05). CONCLUSION: In combination with previous data (unpublished observations), ambulation at G/3 is sufficient to maintain anabolic signaling capacity when compared to G/6 or 0G, suggesting that a threshold level of stimulus is necessary to maintain anabolic capacity in muscle. These results may have important implications towards the development of strategies designed to counter the effects of partial/complete unloading on skeletal muscle based on how the anabolic capacity of muscle is affected.

Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition

We investigated the impact of vitamin D deficiency and repletion on muscle anabolism in old rats. Animals were fed a control (1 IU vitamin D3/g, ctrl, n=20) or a vitamin D-depleted diet (VDD; 0 IU, n=30) for 6 months. A subset was thereafter sacrificed in the control (ctrl6) and depleted groups (VDD6). Remaining control animals were kept for 3 additional months on the same diet (ctrl9), while a part of VDD rats continued on a depleted diet (VDD9) and another part was supplemented with vitamin D (5 IU, VDS9). The ctr16 and VDD6 rats and the ctr19, VDD9 and VDS9 rats were 21 and 24 months old, respectively. Vitamin D status, body weight and composition, muscle strength, weight and lipid content were evaluated. Muscle protein synthesis rate (fractional synthesis rate; FSR) and the activation of controlling pathways were measured. VDD reduced plasma 25(OH)-vitamin D, reaching deficiency (<25 nM), while 25(OH)-vitamin D increased to 118 nM in the VDS group (P<.0001). VDD animals gained weight (P<.05) with no corresponding changes in lean mass or muscle strength. Weight gain was associated with an increase in fat mass (+63%, P<.05), intramyocellular lipids (+75%, P<.05) and a trend toward a decreased plantaris weight (−19%, P=.12). Muscle FSR decreased by 40% in the VDD group (P<.001), but was restored by vitamin D supplementation (+70%, P<.0001). Such changes were linked to an over-phosphorylation of eIF2α. In conclusion, vitamin D deficiency in old rats increases adiposity and leads to reduced muscle protein synthesis through activation of eIF2α. These disorders are restored by vitamin D supplementation.

Influence of omega-3 fatty acids on skeletal muscle protein metabolism and mitochondrial bioenergetics in older adults.

Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults.

Two Weeks of Reduced Steps Disrupts Integrated Rates of Muscle Protein Synthesis and Glycemic Control in Healthy Older Adults

Transient periods of physical inactivity can result in losses of skeletal muscle mass and reductions in insulin sensitivity. These metabolic impairments may not be fully recovered, particularly in older individuals.1 We aimed to examine the impact of two weeks of reduced steps followed by two weeks of recovery, on lean body mass (LBM), integrated rates of myofibrillar protein synthesis (MPS), single fiber cross‐sectional area (fCSA), and indices of glycemic control (HOMA‐IR and MATSUDA) in response to an oral glucose tolerance test. We also examined changes in oxidative phosphorylation protein complex content, phosphorylation of other related signaling molecules, and concentration of circulating inflammatory markers. Twenty‐two healthy older adults (12 men, 10 women, 69±4yr [all data mean±SD]) underwent 7d of monitored normal activity at baseline (BL) before reducing their daily step‐count from 7362±3294 to 990±97 steps.d−1 for 14d (SR). Habitual step count was then recovered for a period of 14d (RC). Integrated rates of muscle protein synthesis (MPS) were reduced from BL at SR (BL 1.51±0.07 to SR 1.33±0.05 %.d−1, p&lt;0.05) and were not restored at RC (1.34±0.14 %.d−1). Women displayed significantly greater rates of integrated MPS at BL compared to men (1.56±0.06 vs. 1.47±0.04 %.d−1, p&lt;0.05). HOMA‐IR increased from BL to SR (2.6±0.4 to 3.8±0.9, p&lt;0.05) and MATSUDA decreased (3.9±0.7 to 2.9±0.4, p&lt;0.05), neither recovered at RC. Glucose AUC was significantly elevated from BL at SR (368±137 to 442±142, p&lt;0.05) and was not fully recovered from SR at RC (393±15). Insulin AUC increased from BL to SR (3372±889 to 3972±1008, p&lt;0.05) and was still elevated at RC (3865±716). Circulating plasma TNF‐a was significantly increased from BL at SR (11.78±2.84 to 15.41±4.64 pg.mL−1, p&lt;0.05) and was still elevated above BL at RC (14.28±3.76 pg.mL−1). Plasma IL‐6 concentration was also significantly increased from BL at SR (8.33±3.06 to 10.84± 4.35 pg.mL−1, p&lt;0.05) and remained elevated at RC (9.99±4.37 pg.mL−1). There was no detectable impact of step reduction on LBM, fCSA, fiber type distribution, or the content of oxidative phosphorylation protein complexes at any time point. Additionally, step reduction failed to impact the phosphorylation status of ACCSer212, AktSer473, mTORSer2448, Ulk1Ser757, and Ulk1Ser55 at BL, SR, and RC. These data demonstrate that two weeks of step reduction leads to lowered rates of MPS that are accompanied by appearance of a diabetic phenotype, and markers of an inflammatory state, which are not recovered on return to a normal daily step count in healthy older adults. Ultimately, proactive interventions such as exercise or pharmaceuticals may be required to reverse decrements in metabolic health with transient periods of physical inactivity in this population.Support or Funding InformationSupported by the Canadian Diabetes Association

Sustained Postprandial Muscle Protein Synthesis Rates after Protein Ingestion in Healthy Young Males

We determined the effect of ingesting a single meal‐like amount of protein on protein digestion and absorption kinetics and the subsequent time‐dependent regulation of postprandial muscle protein synthesis rates in vivo in humans. Seven healthy young males (age: 22±1 y, BMI: 24.8±1.2 kg/m2) received a primed continuous L‐[ring‐2H5]phenylalanine infusions and ingested 38 g intrinsically L‐[1‐13C]phenylalanine‐labeled milk protein concentrate. Biopsies were collected from the vastus lateralis in the basal‐state and 1, 2, 3, and 5 h after protein ingestion. Exogenous phenylalanine appearance in the circulation rapidly increased after protein ingestion (P&lt;0.001). Dietary protein‐derived phenylalanine released in the circulation was 11±2% of the total intake within the first 1 h, followed by 14±1%, 12±1%, and 20±1% within the subsequent 1–2 h, 2–3 h, and 3–5 h of the postprandial period, respectively (total 56±4% over the 5 h period). Myofibrillar protein synthesis rates tended (P=0.10) to increase (absolute change from basal: 0.010±0.008 %/h) immediately following protein ingestion at 0–1 h. Myofibrillar protein synthesis rates were significantly increased at 1–2 h (0.035±0.012 %/h) and myofibrillar protein synthesis remained elevated between 2–3 h (0.019±0.005 %/h) and 3–5 h (0.016±0.004 %/h) after protein ingestion when compared to basal rates (all, P&lt;0.05). Our results show that postprandial muscle protein synthesis rates rapidly increase after protein ingestion and remain elevated above baseline values for at least 5 hours after protein ingestion in healthy young males.Support or Funding InformationIllinois Campus Research Board

Brain Plasticity: Measuring Protein Synthesis Rates in the Human Brain

IntroductionBrain plasticity is a term typically used to refer to the ability of the brain to reorganize itself throughout life. Though the term is often applied in neuroscience to express synaptic or non‐synaptic plasticity, information on whether neuroplasticity is also accompanied by a high brain tissue protein turnover rate is scarce. Because of obvious limitations with regard to brain tissue sampling no study has ever directly measured brain protein synthesis rates in vivo in humans. In this study, we applied contemporary stable isotope methodology to assess protein synthesis rates in vivo in human brain tissue of patients operated for drug‐resistant epilepsy.MethodsSix otherwise healthy patients (47±6 y), scheduled to undergo resective surgery for treatment of drug‐resistant temporomesial epilepsy, were included in this study. Primed continuous intravenous infusions with L‐[ring‐13C6]‐Phenylalanine and L‐[3,5‐2H2]‐Tyrosine were initiated 2.5 h prior to surgery and continued during surgery. Throughout the surgical procedure the following tissue samples were obtained: temporal neocortex, hippocampus, temporalis muscle, and vastus lateralis muscle. Tissue‐specific fractional protein synthesis rates (%/h) were assessed by measuring the incorporation of labelled L‐[ring‐13C6]‐Phenylalanine in tissue protein and were compared between the different tissues using a paired t test.ResultsSerum L‐[ring‐13C6]‐Phenylalanine enrichments averaged 8.1±0.7 MPE throughout the surgical procedure. Protein synthesis rates of temporal neocortex and hippocampus tissue averaged 0.17±0.01 and 0.13±0.01 %/h, respectively, with a significant difference between both tissues (P&lt;0.05). Brain tissue protein synthesis rates were 3–4 fold higher compared to skeletal muscle protein synthesis rates (0.05±0.01 %/h; P&lt;0.001).ConclusionThis is the first study to present protein synthesis rates in vivo in the human brain. With brain protein synthesis rates being several‐fold higher compared to skeletal muscle protein synthesis rates, the human brain displays a degree of tissue plasticity much higher than generally assumed.

Energy and protein requirements for growth of Holstein×Gyr heifers.

There is little information regarding the nutritional requirements for dairy heifers, leading the majority of nutrient requirement systems to consider dairy heifers to be similar to beef heifers. Therefore, we evaluated the muscle protein metabolism and physical and chemical body composition of growing Holstein×Gyr heifers and estimated the energy and protein requirements. We performed a comparative slaughter experiment with 20 Holstein×Gyr heifers at an initial body weight of 218±36.5kg and an average age of 12±1.0months. Four heifers were designated as the reference group, and the 16 remaining heifers were fed adlibitum. The 16 heifers were distributed using a completely randomized design in a 2×2 factorial arrangement with two roughages (corn silage or sugarcane) and two concentrate levels (30 or 50%) for 112days. Greater (p<0.05) values for fractional rates of muscle protein synthesis, degradation and accretion were observed for heifers that were fed 50% concentrate. The following equations were obtained to estimate the net energy for gain (NEg ) and net protein for gain (NPg ): NEg (Mcal/day)=0.0685×EBW0.75 ×EBWG1.095 and NPg (g/day)=203.8× EBWG-14.80×RE, respectively, in which EBW is the empty body weight, EBWG is the empty body weight gain and RE is the retained energy. We concluded that increased rates of protein turnover are achieved when a greater quality diet is provided. In the future, these results can be used to calculate the nutritional requirements for growth of Holstein×Gyr heifers after equationvalidation rather than using the recommendations provided by other systems, which use values developed from beef heifers, to determine the nutritional requirements of dairy cattle.

Co-ingestion of carbohydrate and whey protein increases fasted rates of muscle protein synthesis immediately after resistance exercise in rats.

The objective of the study was to investigate whether co-ingestion of carbohydrate and protein as compared with protein alone augments muscle protein synthesis (MPS) during early exercise recovery. Two months old rats performed 10 repetitions of ladder climbing with 75% of body weight attached to their tails. Placebo (PLA), whey protein (WP), or whey protein plus carbohydrate (CP) was then given to rats by gavage. An additional group of sedentary rats (SED) was used as controls. Blood samples were collected immediately and at either 1 or 2 h after exercise. The flexor hallucis longus muscle was excised at 1 or 2 h post exercise for analysis of MPS and related signaling proteins. MPS was significantly increased by CP compared with PLA (p<0.05), and approached significance compared with WP at 1 h post exercise (p = 0.08). CP yielded a greater phosphorylation of mTOR compared with SED and PLA at 1 h post exercise and SED and WP at 2 h post exercise. CP also increased phosphorylation of p70S6K compared with SED at 1 and 2 h post exercise. 4E-BP1 phosphorylation was inhibited by PLA at 1 h but elevated by WP and CP at 2 h post exercise relative to SED. The phosphorylation of AMPK was elevated by exercise at 1 h post exercise, and this elevated level was sustained only in the WP group at 2 h. The phosphorylation of Akt, GSK3, and eIF2Bε were unchanged by treatments. Plasma insulin was transiently increased by CP at 1 h post exercise. In conclusion, post-exercise CP supplementation increases MPS post exercise relative to PLA and possibly WP, which may have been mediated by greater activation of the mTOR signaling pathway.

Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6weeks of milk protein feeding in older adults.

In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6weeks of milk protein feeding in older adults.

11C-L-methyl methionine dynamic PET/CT of skeletal muscle: response to protein supplementation compared to L-[ring 13C6] phenylalanine infusion with serial muscle biopsy

ObjectiveThe objective of this study was to determine if clinical dynamic PET/CT imaging with 11C-L-methyl-methionine (11C-MET) in healthy older women can provide an estimate of tissue-level post-absorptive and post-prandial skeletal muscle protein synthesis that is consistent with the more traditional method of calculating fractional synthesis rate (FSR) of muscle protein synthesis from skeletal muscle biopsies obtained during an infusion of L-[ring 13C6] phenylalanine (13C6-Phe).MethodsHealthy older women (73 ± 5 years) completed both dynamic PET/CT imaging with 11C-MET and a stable isotope infusion of 13C6-Phe with biopsies to measure the skeletal muscle protein synthetic response to 25 g of a whey protein supplement. Graphical estimation of the Patlak coefficient Ki from analysis of the dynamic PET/CT images was employed as a measure of incorporation of 11 C-MET in the mid-thigh muscle bundle.ResultsPost-prandial values [mean ± standard error of the mean (SEM)] were higher than post-absorptive values for both Ki (0.0095 ± 0.001 vs. 0.00785 ± 0.001 min−1, p < 0.05) and FSR (0.083 ± 0.008 vs. 0.049 ± 0.006%/h, p < 0.001) in response to the whey protein supplement. The percent increase in Ki and FSR in response to the whey protein supplement was significantly correlated (r = 0.79, p = 0.015).ConclusionsDynamic PET/CT imaging with 11C-MET provides an estimate of the post-prandial anabolic response that is consistent with a traditional, invasive stable isotope, and muscle biopsy approach. These results support the potential future use of 11C-MET imaging as a non-invasive method for assessing conditions affecting skeletal muscle protein synthesis.

Habituation to low or high protein intake does not modulate basal or postprandial muscle protein synthesis rates: a randomized trial

Background: Muscle mass maintenance is largely regulated by basal muscle protein synthesis rates and the ability to increase muscle protein synthesis after protein ingestion. To our knowledge, no previous studies have evaluated the impact of habituation to either low protein intake (LOW PRO) or high protein intake (HIGH PRO) on the postprandial muscle protein synthetic response.Objective: We assessed the impact of LOW PRO compared with HIGH PRO on basal and postprandial muscle protein synthesis rates after the ingestion of 25 g whey protein.Design: Twenty-four healthy, older men [age: 62 ± 1 y; body mass index (in kg/m2): 25.9 ± 0.4 (mean ± SEM)] participated in a parallel-group randomized trial in which they adapted to either a LOW PRO diet (0.7 g · kg–1 · d−1; n = 12) or a HIGH PRO diet (1.5 g · kg–1 · d–1; n = 12) for 14 d. On day 15, participants received primed continuous l-[ring-2H5]-phenylalanine and l-[1-13C]-leucine infusions and ingested 25 g intrinsically l-[1-13C]-phenylalanine– and l-[1-13C]-leucine–labeled whey protein. Muscle biopsies and blood samples were collected to assess muscle protein synthesis rates as well as dietary protein digestion and absorption kinetics.Results: Plasma leucine concentrations and exogenous phenylalanine appearance rates increased after protein ingestion (P < 0.01) with no differences between treatments (P > 0.05). Plasma exogenous phenylalanine availability over the 5-h postprandial period was greater after LOW PRO than after HIGH PRO (61% ± 1% compared with 56% ± 2%, respectively; P < 0.05). Muscle protein synthesis rates increased from 0.031% ± 0.004% compared with 0.039% ± 0.007%/h in the fasted state to 0.062% ± 0.005% compared with 0.057% ± 0.005%/h in the postprandial state after LOW PRO compared with HIGH PRO, respectively (P < 0.01), with no differences between treatments (P = 0.25).Conclusion: Habituation to LOW PRO (0.7 g · kg–1 · d–1) compared with HIGH PRO (1.5 g · kg–1 · d–1) augments the postprandial availability of dietary protein–derived amino acids in the circulation and does not lower basal muscle protein synthesis rates or increase postprandial muscle protein synthesis rates after ingestion of 25 g protein in older men. This trial was registered at clinicaltrials.gov as NCT01986842.

Precocious glucocorticoid exposure reduces skeletal muscle satellite cells in the fetal rat.

Perinatal skeletal muscle growth rates are a function of protein and myonuclear accretion. Precocious exposure of the fetus to glucocorticoids (GLC) in utero impairs muscle growth. Reduced muscle protein synthesis rates contribute to this response, but the consequences for myonuclear hyperplasia are unknown. To test the hypothesis that blunting of Pax7+ muscle progenitor cell proliferative activity by GLC in vivo also contributes to reduced fetal muscle growth, pregnant rats were administered dexamethasone (DEX: 1 mg/L drinking water) from embryonic day (ED) 13 to ED21. Their responses were compared to pair-fed (PF) and ad libitum-fed controls (CON). Bromodeoxyuridine (BrdU) was administered before delivery to measure myonuclear accretion. Fetal hind limb and diaphragm muscles were collected at term and analyzed for myofiber cross-sectional area (CSA), total and BrdU+ myonuclei, Pax7+ nuclei, MyoD and myogenin protein and mRNA abundance and myosin heavy chain (MyHC) isoform composition. Mean fiber CSA, myonuclei/myofiber and Pax7+ nuclei/myofiber ratios were reduced in DEX compared to those in CON and PF muscles; CSA/myonucleus, BrdU+/total myonuclei and BrdU+ myonuclei/Pax7+ nuclei were similar among groups. Myogenin abundance was reduced and MyHC-slow was increased in DEX fetuses. The data are consistent with GLC inhibition of muscle progenitor cell proliferation limiting satellite cell and myonuclear accretion. The response of PF-fed compared to CON muscles indicated that decreased food consumption by DEX dams contributed to the smaller myofiber CSA but did not affect Pax7+ nuclear accretion. Thus, the effect on satellite cell reserve and myonuclear number also contributes to the blunting of fetal muscle growth by GLC.

Native whey protein with high levels of leucine results in similar post-exercise muscular anabolic responses as regular whey protein: a randomized controlled trial

BackgroundProtein intake is essential to maximally stimulate muscle protein synthesis, and the amino acid leucine seems to possess a superior effect on muscle protein synthesis compared to other amino acids. Native whey has higher leucine content and thus a potentially greater anabolic effect on muscle than regular whey (WPC-80). This study compared the acute anabolic effects of ingesting 2 × 20 g of native whey protein, WPC-80 or milk protein after a resistance exercise session.MethodsA total of 24 young resistance trained men and women took part in this double blind, randomized, partial crossover, controlled study. Participants received either WPC-80 and native whey (n = 10), in a crossover design, or milk (n = 12). Supplements were ingested immediately (20 g) and two hours after (20 g) a bout of heavy-load lower body resistance exercise. Blood samples and muscle biopsies were collected to measure plasma concentrations of amino acids by gas-chromatography mass spectrometry, muscle phosphorylation of p70S6K, 4E–BP1 and eEF-2 by immunoblotting, and mixed muscle protein synthesis by use of [2H5]phenylalanine-infusion, gas-chromatography mass spectrometry and isotope-ratio mass spectrometry. Being the main comparison, differences between native whey and WPC-80 were analysed by a one-way ANOVA and comparisons between the whey supplements and milk were analysed by a two-way ANOVA.ResultsNative whey increased blood leucine concentrations more than WPC-80 and milk (P < 0.05). Native whey ingestion induced a greater phosphorylation of p70S6K than milk 180 min after exercise (P = 0.03). Muscle protein synthesis rates increased 1–3 h hours after exercise with WPC-80 (0.119%), and 1–5 h after exercise with native whey (0.112%). Muscle protein synthesis rates were higher 1–5 h after exercise with native whey than with milk (0.112% vs. 0.064, P = 0.023).ConclusionsDespite higher-magnitude increases in blood leucine concentrations with native whey, it was not superior to WPC-80 concerning effect on muscle protein synthesis and phosphorylation of p70S6K during a 5-h post-exercise period. Native whey increased phosphorylation of p70S6K and muscle protein synthesis rates to a greater extent than milk during the 5-h post exercise period.Trial registrationThis study was retrospectively registered at clinicaltrials.gov as NCT02968888.

Effects of an amylopectin and chromium complex on the anabolic response to a suboptimal dose of whey protein

BackgroundPrevious research has demonstrated the permissive effect of insulin on muscle protein kinetics, and the enhanced insulin sensitizing effect of chromium. In the presence of adequate whole protein and/or essential amino acids (EAA), insulin has a stimulatory effect on muscle protein synthesis, whereas in conditions of lower blood EAA concentrations, insulin has an inhibitory effect on protein breakdown. In this study, we determined the effect of an amylopectin/chromium (ACr) complex on changes in plasma concentrations of EAA, insulin, glucose, and the fractional rate of muscle protein synthesis (FSR).MethodsUsing a double-blind, cross-over design, ten subjects (six men, four women) consumed 6 g whey protein + 2 g of the amylopectin-chromium complex (WPACr) or 6 g whey protein (WP) after an overnight fast. FSR was measured using a primed, continuous infusion of ring-d5-phenylalanine with serial muscle biopsies performed at 2, 4, and 8 h. Plasma EAA and insulin were assayed by ion-exchange chromatography and ELISA, respectively. After the biopsy at 4 h, subjects ingested their respective supplement, completed eight sets of bilateral isotonic leg extensions at 80% of their estimated 1-RM, and a final biopsy was obtained 4 h later.ResultsBoth trials increased EAA similarly, with peak levels noted 30 min after ingestion. Insulin tended (p = 0.09) to be higher in the WPACr trial. Paired samples t-tests using baseline and 4-h post-ingestion FSR data separately for each group revealed significant increases in the WPACr group (+0.0197%/h, p = 0.0004) and no difference in the WP group (+0.01215%/hr, p = 0.23). Independent t-tests confirmed significant (p = 0.045) differences in post-treatment FSR between trials.ConclusionsThese data indicate that the addition of ACr to a 6 g dose of whey protein (WPACr) increases the FSR response beyond what is seen with a suboptimal dose of whey protein alone.

The Effect of Casein Ingestion within a Milk Matrix on Muscle Protein Synthesis

Isolated micellar casein has been classified as a slow protein because of its slow digestion and amino acid absorption kinetics. These result further in a more moderate and sustained amino acid availability in the plasma and subsequently lower muscle protein synthesis (MPS) rate when, for example, compared with whey. However, the milk matrix with lactose, fat and various minerals may modulate the effect of casein on MPS. Therefore, this study aimed to compare the effects of the ingestion of casein dissolved in bovine milk serum to the ingestion of isolated casein on myofibrillar protein synthesis. In this parallel group randomized trial, 32 healthy older men (age: 71 ± 1 y) received a primed continuous infusion of L-[ring-2 H5 ]phenylalanine, L-[1-13C]leucine, and L-[ring-3,5-2 H2 ]tyrosine and blood and muscle samples were taken to assess myofibrillar fractional synthetic rate (FSR) under basal postabsorptive conditions and after a single bolus of 25 g intrinsically L-[1-13C]phenylalanine and L-[1-13C]leucine-labeled casein either in an isolated form (ISOCAS; n = 16) or dissolved in bovine milk serum (MILK-CAS; n = 16). The ingestion of 25 g of casein significantly (P &lt; 0.05) increased MPS rates in both groups when assessed over the late postprandial period (t = 120 – 300 min). Ingestion of MILK-CAS and ISO-CAS did not significantly stimulate MPS rates when assessed over the early (t = 0 – 120 min) and overall (t = 0 – 300 min) postprandial FSR compared to postabsorptive (t = -120 – 0 min) FSR. No significant differences were observed between the two groups over the early, late or entire postprandial period (P &gt; 0.05). The ingestion of additional normal milk matrix to micellar casein does not modulate overall myofibrillar protein synthesis rates in older men when compared to the ingestion of micellar casein dissolved in water. Therefore, there is neither a benefit, nor a detrimental effect of ingesting micellar casein within a normal milk matrix instead of the isolated form to increase MPS.

Resistance Exercise Augments Postprandial Overnight Muscle Protein Synthesis Rates.

We have previously shown that protein ingestion before sleep increases overnight muscle protein synthesis rates. Whether prior exercise further augments the muscle protein synthetic response to presleep protein ingestion remains to be established. This study aimed to assess whether resistance-type exercise performed in the evening increases the overnight muscle protein synthetic response to presleep protein ingestion. Twenty-four healthy young men were randomly assigned to ingest 30 g intrinsically L-[1-C]-phenylalanine and L-[1-C]-leucine-labeled casein protein before going to sleep with (PRO + EX, n = 12) or without (PRO, n = 12) prior resistance-type exercise performed in the evening. Continuous intravenous L-[ring-H5]-phenylalanine, L-[1-C]-leucine, and L-[ring-H2]-tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole-body protein balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into de novo myofibrillar protein. A total of 57% ± 1% of the ingested protein-derived phenylalanine appeared in the circulation during overnight sleep. Overnight myofibrillar protein synthesis rates were 37% (0.055%·h ± 0.002%·h vs. 0.040%·h ± 0.003%·h, P < 0.001, based on L-[ring- H5]-phenylalanine) and 31% (0.073%·h ± 0.004%·h vs. 0.055%·h ± 0.006%·h, P = 0.024, based on L-[1-C]-leucine) higher in PRO + EX compared with PRO. Substantially more of the dietary protein-derived amino acids were incorporated into de novo myofibrillar protein during overnight sleep in PRO + EX compared with PRO (0.026 ± 0.003 vs. 0.015 ± 0.003 molar percent excess, P = 0.012). Resistance-type exercise performed in the evening augments the overnight muscle protein synthetic response to presleep protein ingestion and allows more of the ingested protein-derived amino acids to be used for de novo myofibrillar protein synthesis during overnight sleep.

Pre-Sleep Protein Ingestion to Improve the Skeletal Muscle Adaptive Response to Exercise Training.

Protein ingestion following resistance-type exercise stimulates muscle protein synthesis rates, and enhances the skeletal muscle adaptive response to prolonged resistance-type exercise training. As the adaptive response to a single bout of resistance exercise extends well beyond the first couple of hours of post-exercise recovery, recent studies have begun to investigate the impact of the timing and distribution of protein ingestion during more prolonged recovery periods. Recent work has shown that overnight muscle protein synthesis rates are restricted by the level of amino acid availability. Protein ingested prior to sleep is effectively digested and absorbed, and thereby stimulates muscle protein synthesis rates during overnight recovery. When applied during a prolonged period of resistance-type exercise training, protein supplementation prior to sleep can further augment gains in muscle mass and strength. Recent studies investigating the impact of pre-sleep protein ingestion suggest that at least 40 g of protein is required to display a robust increase in muscle protein synthesis rates throughout overnight sleep. Furthermore, prior exercise allows more of the pre-sleep protein-derived amino acids to be utilized for de novo muscle protein synthesis during sleep. In short, pre-sleep protein ingestion represents an effective dietary strategy to improve overnight muscle protein synthesis, thereby improving the skeletal muscle adaptive response to exercise training.

Marginal dietary zinc deprivation augments sepsis-induced alterations in skeletal muscle TNF-α but not protein synthesis.

Severe zinc deficiency is associated with an increased systemic inflammatory response and mortality after sepsis. However, the impact of mild zinc deficiency, which is more common in populations with chronic illnesses and sepsis, is unknown. In this study, we hypothesized that marginal dietary Zn deprivation (ZM) would amplify tissue inflammation and exacerbate the sepsis‐induced decrease in muscle protein synthesis. Adult male C57BL/6 mice were fed a zinc‐adequate (ZA) or ZM diet (30 or 10 mg Zn/kg, respectively) over 4 weeks, peritonitis was induced by cecal ligation and puncture (CLP), and mice were examined at either 24 h (acute) or 5 days (chronic) post‐CLP. Acute sepsis decreased the in vivo rate of skeletal muscle protein synthesis and the phosphorylation of the mTOR substrate 4E‐BP1. Acutely, sepsis increased TNF‐α and IL‐6 mRNA in muscle, and the increase in TNF‐α was significantly greater in ZM mice. However, muscle protein synthesis and 4E‐BP1 phosphorylation returned to baseline 5 days post‐CLP in both ZA and ZM mice. Protein degradation via markers of the ubiquitin proteasome pathway was increased in acute sepsis, yet only MuRF1 mRNA was increased in chronic sepsis and ZM amplified this elevation. Our data suggest that mild zinc deficiency increases TNF‐α in muscle acutely after sepsis but does not significantly modulate the rate of muscle protein synthesis.