Fractional Synthesis Research Articles

Beta2 -adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men.

Animal models have shown that beta2 -adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2 -adrenoceptor is a potential target in the treatment of disuse-, disease- and age-related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2 -adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A-signalling, activation of Akt2 and modulation of mRNA levels of growth-regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2 -adrenoceptor agonist treatment during recovery from resistance exercise in humans. The effect of beta2 -adrenoceptor stimulation on skeletal muscle protein turnover and intracellular signalling is insufficiently explored in humans, particularly in association with exercise. In a randomized, placebo-controlled, cross-over study investigating 12 trained men, the effects of beta2 -agonist (6×4mg oral salbutamol) on protein turnover rates, intracellular signalling and mRNA response in skeletal muscle were investigated 0.5-5h after quadriceps resistance exercise. Each trial was preceded by a 4-day lead-in treatment period. Leg protein turnover rates were assessed by infusion of [13 C6 ]-phenylalanine and sampling of arterial and venous blood, as well as vastus lateralis muscle biopsies 0.5 and 5h after exercise. Furthermore, myofibrillar fractional synthesis rate, intracellular signalling and mRNA response were measured in muscle biopsies. The mean (95% confidence interval) myofibrillar fractional synthesis rate was higher for salbutamol than placebo [0.079(95% CI, 0.064 to 0.093) vs. 0.066(95% CI, 0.056 to 0.075%)×h-1 ] (P<0.05). Mean net leg phenylalanine balance 0.5-5h after exercise was higher for salbutamol than placebo [3.6(95% CI, 1.0 to 6.2nmol)×min-1 ×100 gLeg Lean Mass-1 ] (P<0.01). Phosphorylation of Akt2, cAMP response element binding protein and PKA substrate 0.5 and 5h after exercise, as well as phosphorylation of eEF2 5h after exercise, was higher (P<0.05) for salbutamol than placebo. Calpain-1, Forkhead box protein O1, myostatin and Smad3 mRNA content was higher (P<0.01) for salbutamol than placebo 0.5h after exercise, as well as Forkhead box protein O1 and myostatin mRNA content 5h after exercise, whereas ActivinRIIB mRNA content was lower (P<0.01) for salbutamol 5h after exercise. These observations suggest that beta2 -agonist increases protein turnover rates in skeletal muscle after resistance exercise in humans, with concomitant cAMP/PKA and Akt2 signalling, as well as modulation of mRNA response of growth-regulating proteins.

DDFS and ΣΔ Approaches for Fractional Frequency Synthesis in Terahertz Instruments

This paper compares direct digital frequency synthesis (DDFS) and ΣΔ CMOS-based frequency synthesizers for terahertz instrument systems. This paper first discusses the need for fractional synthesis in terahertz systems, and provides a brief overview of reference clock frequency selection, and design choices related to implementation of fractional functionality. ΣΔ-multistage-noise-shape-based and DDFS-based fractional synthesis approaches and architectures are discussed and CMOS synthesizer modules based on both of these approaches are presented. Finally, the paper compares two different 100-GHz CMOS fractional frequency synthesizer chips, which employ the same millimeter-wave circuits but different fractional generators (third- and sixth-order ΣΔ, and DDFS-based). Performance in terms of phase-noise, spurious components, and power consumption are directly compared. Measurements suggest ΣΔ synthesis is more suitable than DDFS for THz fractional synthesizers due to the high-phase multiplication of spurs.

Glutathione metabolism in type 2 diabetes and its relationship with microvascular complications and glycemia.

Aims/HypothesesWe hypothesized that there is decreased synthesis of glutathione (GSH) in type 2 diabetes (T2DM) especially in the presence of microvascular complications, and this is dependent on the degree of hyperglycemia.MethodsIn this case-control study, we recruited 16 patients with T2DM (7 without and 9 with microvascular complications), and 8 age- and sex-matched non-diabetic controls. We measured GSH synthesis rate using an infusion of [2H2]-glycine as isotopic tracer and collection of blood samples for liquid chromatography mass spectrometric analysis.ResultsCompared to the controls, T2DM patients had lower erythrocyte GSH concentrations (0.90 ± 0.42 vs. 0.35 ± 0.30 mmol/L; P = 0.001) and absolute synthesis rates (1.03 ± 0.55 vs. 0.50 ± 0.69 mmol/L/day; P = 0.01), but not fractional synthesis rates (114 ± 45 vs. 143 ± 82%/day; P = 0.07). The magnitudes of changes in patients with complications were greater for both GSH concentrations and absolute synthesis rates (P-values ≤ 0.01) compared to controls. There were no differences in GSH concentrations and synthesis rates between T2DM patients with and without complications (P-values > 0.1). Fasting glucose and HbA1c did not correlate with GSH concentration or synthesis rates (P-values > 0.17).ConclusionsCompared to non-diabetic controls, patients with T2DM have glutathione deficiency, especially if they have microvascular complications. This is probably due to reduced synthesis and increased irreversible utilization by non-glycemic mechanisms.

Determinants of skeletal muscle protein turnover following severe burn trauma in children

Burns remain the fifth cause of non-fatal pediatric injuries globally, with muscle cachexia being a hallmark of the stress response to burns. Burn-induced muscle wasting is associated with morbidity, yet the determinants of muscle protein catabolism in response to burn trauma remains unclear. Our objective was to determine the effect of patient and injury characteristics on muscle protein kinetics in burn patients. This retrospective, observational study was performed using protein kinetic data from pediatric patients who had severe burns (>30% of the total body surface area burned) and underwent cross-limb stable isotope infusions between 1999 and 2008 as part of prospective clinical trials. Mixed multiple regression models were used to assess associations between patient/injury characteristics and muscle protein fractional synthesis rate (FSR), net balance (NB), and rates of phenylalanine appearance (Ra; index of protein breakdown) and disappearance (Rd; index of protein synthesis) across the leg. A total of 268 patients who underwent 499 studies were analyzed. Increasing time post injury was associated with greater FSR (p<0.001) and NB (p=0.01). Males were more catabolic than females (as indicated by lower NB, p=0.04 and greater Ra, p=0.008), a consequence of higher protein breakdown rather than lower synthesis. Increasing burn size was associated with higher protein synthesis rate (as indicated by higher FSR, p=0.019) and higher protein breakdown rates (as indicated by greater Ra, p=0.001). FSR was negatively associated with age (p<0.001). Data from this large patient cohort show that injury severity, sex, and time post injury influence skeletal muscle wasting in burned children. These findings suggest that individual patient characteristics should be considered when devising therapies to improve the acute care and rehabilitation of burn survivors.

Whole-body nitrogen utilization and tissue protein and casein synthesis in lactating primiparous sows fed low- and high-protein diets.

Twenty-eight lactating Yorkshire and Yorkshire × Landrace primiparous sows were used to test the hypothesis that feeding a diet with reduced CP concentration and supplemented with crystalline AA (CAA) does not decrease milk protein yield and litter growth but improves apparent N utilization for milk protein production. Sows were assigned to 1 of 2 dietary treatments: 1) control (CON; 16.2% CP; analyzed content) or 2) low CP with CAA to meet estimated requirements of limiting AA (LCP; 12.7% CP) over a 17-d lactation period. A N balance was conducted for each sow between days 13 and 17 of lactation. On day 17, a 12-h primed continuous infusion of l-[ring-2H5]-Phe was conducted on 12 sows (n = 6) with serial blood and milk sampling to determine plasma AA concentrations and Phe enrichment, and milk casein synthesis, respectively. Thereafter, sows were sacrificed and tissues were collected to determine tissue protein fractional synthesis rates (FSR). Litter growth rate and milk composition did not differ. Sows fed the LCP diet had reduced N intake (122.7 vs. 153.2 g/d; P < 0.001) and maternal N retention (13.5 vs. 24.6 g/d; P < 0.05) and greater apparent efficiency of using dietary N intake for milk production (85.1% vs. 67.5%; P < 0.001). On day 17 of lactation, all plasma essential AA concentrations exhibited a quartic relationship over time relative to consumption of a meal, where peaks occurred at approximately 1- and 4-h postprandial (P < 0.05). Protein FSR in liver, LM, gastrocnemius muscle, mammary gland, and in milk caseins did not differ between treatments. Feeding primiparous sows with a diet containing 12.7% CP and supplemented with CAA to meet the limiting AA requirements did not reduce milk protein yield or piglet growth rate and increased the apparent utilization of dietary N, Arg, Leu, Phe+Tyr, and Trp for milk protein production. The improved apparent utilization of N and AA appears to be related exclusively to a reduction in N and AA intake.

Fast and exact synthesis of some operator scaling Gaussian random fields

Abstract Operator scaling Gaussian random fields, as anisotropic generalizations of self-similar fields, know an increasing interest for theoretical studies in the literature. However, up to now, they were only defined through stochastic integrals, without explicit covariance functions. In this paper we exhibit explicit covariance functions, as anisotropic generalizations of fractional Brownian fields ones, and define corresponding Operator scaling Gaussian random fields. This allows us to propose a fast and exact method of simulation in dimension 2 based on the circulant embedding matrix method, following ideas of Stein [34] for fractional Brownian surfaces syntheses. This is a first piece of work to popularize these models in anisotropic spatial data modeling.

The effect of high fat diet and metformin treatment on liver lipids accumulation and their impact on insulin action

We sought to determine whether metformin treatment reverses a high-fat diet (HFD)-induced hepatic insulin resistance (IRes) and to identify lipid intermediates involved in induction of liver IRes. The experiments were conducted on male Wistar rats divided into three groups: 1. Control, 2. fed HFD and 3. fed HFD and treated with metformin. The animals were infused with a [U-13C]palmitate to measure fractional lipid synthesis rate. This allowed for the calculation of fractional synthesis rate of signaling lipids (FSR) through the estimation of their isotopic enrichment. Liver ceramide (Cer), diacylglycerol (DAG) and acyl-carnitine concentration and enrichment were analyzed by LC/MS/MS. The content of proteins involved in lipid metabolism and insulin signaling were analyzed by Western Blot. HFD treatment increased the content and FSR of DAG and Cer in the liver which was accompanied by systemic insulin resistance and inhibition of hepatic insulin signaling pathway under insulin stimulation. Metformin treatment ameliorated systemic insulin resistance and augmented the hepatic insulin signaling cascade. It reduced both the concentration and FSR of Cer, DAG, and increased acyl-carnitine content and the expression of mitochondrial markers. We postulate, that in liver, the insulin sensitizing effect of metformin depends on augmentation of mitochondrial β-oxidation, which protects from hepatic accumulation of both the Cer and DAG and preserves insulin sensitivity under HFD consumption. Moreover, we showed that hepatic content of Cer and DAG corresponds with their respective FSR.

Protein Synthesis throughout the Progression of Cancer Cachexia in Tumor‐Bearing Mice

Cancer cachexia occurs in approximately 80 percent of cancer patients and is a key contributor to cancer‐related death. The mechanisms controlling development of tumor‐induced muscle wasting are not fully elucidated. Specifically, the progression of cancer cachexia is underexplored. We have previously reported that muscle wasting occurs 4 wks following tumor implantation in these mice.PurposeTo examine the rate of muscle protein synthesis throughout the progression of cancer cachexia in tumor‐bearing mice.MethodsLewis Lung Carcinoma (LLC) was injected into the hind‐flank of C57BL6/J mice at 8 wks age with tumor allowed to develop for 1, 2, 3, or 4 wks and compared to PBS injected control. PBS control animals were age matched to the 4 wk tumor bearing group. Cumulative fractional protein synthetic rates (FSR) were determined using deuterium oxide (2H20). Briefly, a (20 μL/g body weight) bolus priming dose of deuterium oxide was injected intraperitoneally approximately 24 hours before tissue collection. Drinking water was thereafter supplemented with 4% deuterium oxide in order to maintain the plasma pool of deuterium oxide. Fractional synthetic rate (FSR) of mixed muscle fraction (soluble, mitochondria, sarcolemmal, and contractile) and myofibrillar (contractile) rich fraction from gastrocnemius muscle were measured us a GC/MS. Data were analyzed by one‐way ANOVA. When significant F‐ratios were found, a SNK post hoc was used to compare differences between experimental groups. Significance was established at p&lt;0.05.ResultsPlasma enrichment of 2H20 for all groups was 2.91 ± 0.073% and no significant differences existed in means between groups. Mixed muscle FSR remained consistent in 1 wk and 2 wk animals and was ~40% lower 4 wks following tumor implantation (p&lt;0.05) when compared to PBS control. The myofibrillar rich fraction of FSR followed a similar pattern, but changes did not reach statistical significance between groups.ConclusionsThese data indicate muscle protein synthesis decreases concomitantly with muscle loss and may be a major contributor of cancer cachexia in tumor‐bearing mice. More research needs to be conducted to analyze the mechanism for the loss of protein synthesis in cachectic mice.Support or Funding InformationThis study was supported by The Arkansas Bioscience Institute and National Institutes of Health R15AR069913.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Healthspan‐Extending Treatments Enhance Proteostasis through Reduced Cellular Proliferation

Aging is characterized by an accumulation of damaged intracellular components over time. Mechanisms that can prevent or delay the accumulation of intracellular damage may increase healthspan (i.e., the period of life spent free from chronic disease). Multiple long‐lived models demonstrate an increased ability to maintain protein homeostasis (proteostasis) over the duration of the lifespan. Rapamycin (Rap), and Metformin in combination with Rap (MetRap), have both been demonstrated to increase median lifespan. It is currently unknown how Rap and MetRap influence cellular proteostasis. Thus, the purpose of this study was to examine the influence of the healthspan‐extending treatments Rap and MetRap on protein turnover and cellular proliferation in vitro. We also sought to determine how autophagic flux contributes to protein turnover during Rap and MetRap treatments. We hypothesized that both Rap and MetRap would increase protein turnover (both synthesis and breadown) and reduce cellular proliferation. We further hypothesized that a decrease in autophagic flux would decrease protein turnover during Rap and MetRap treatments. Protein and DNA synthesis or breakdown rates were measured in C2C12 myotubes for up to 24h using deuterium oxide. Rapamycin (Rap 5 nM) and Rap in combination with Metformin (MetRap 2 mM/5 nM) were applied to C2C12 myotubes. Bafilomycin A1 (Baf 100 nM) was used to pre‐ and co‐treat myotubes to block autophagic flux during treatments. Fractional synthesis and breakdown rates were calculated for mitochondrial‐enriched, cytoplasmic‐enriched, and DNA fractions from each 100 mm culture dish in triplicate. Protein/DNA ratios (Pro/DNA) were calculated by dividing the fractional synthesis rate of proteins by DNA fractional synthesis rate to differentiate protein synthesis for growth versus maintaining proteostasis. In accordance with our hypotheses, we found that 24h of Rap treatment significantly increased mitochondrial (P&lt;0.01) and cytoplasmic (P&lt;0.01) Pro/DNA compared to Con. Inhibition of autophagic flux reduced mitochondrial protein breakdown rate by 29% compared to CON (P&lt;0.001), while cytoplasmic protein breakdown was only reduced by 9% compared to CON (P&lt;0.05). Mitochondrial Pro/DNA during Rap and MetRap was not altered by co‐treatment with Baf. However, Baf+Rap significantly increased (P&lt;0.0001) cytoplasmic Pro/DNA compared to Rap as well as compared to CON (P&lt;0.0001). Further, Baf+MetRap significantly increased (P&lt;0.0001) cytoplasmic Pro/DNA compared to MetRap. Baf+Rap and Baf+MetRap significantly reduced cellular DNA synthesis compared to CON, Rap, and MetRap (all P&lt;0.001). Overall, these data suggest that Rap and MetRap improve mitochondrial and cytoplasmic proteostatic mechanisms in vitro, and surprisingly, inhibition of autophagy did not have a negative impact on cytoplasmic proteostatic mechanisms.Support or Funding InformationThis project is supported by NIH‐NIA‐AG‐042569 (BFM and KLH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metformin Blunts Exercise‐Induced Improvements in Skeletal Muscle Mitochondrial Respiration Independent of Changes in Mitochondrial Biogenesis

The anti‐hyperglycemic agent Metformin and exercise independently prevent the progression from prediabetes to overt type two diabetes (T2D) and are both implicated in healthspan extension. However, emerging evidence indicates the addition of Metformin to exercise may inhibit the health benefits of exercise. The purpose of the current study was to test the hypothesis that Metformin blunts the positive adaptations to exercise by inhibiting skeletal muscle mitochondrial fractional protein synthesis rates (FSR) and respiration. Older adults (n=53, 62±5 yrs) with at least one risk factor for T2D were randomized in a double‐blinded fashion to consume Metformin or placebo (1500–2000 mg/day) during 12‐weeks of aerobic exercise training (AET). VO2max and skeletal muscle mitochondrial respiration were assessed before (PRE) and after (POST) the 12‐week intervention. Skeletal muscle biopsy samples were obtained 48 hrs after the last exercise bout and 36 hrs after the last metformin dose. High‐resolution respirometry was performed on permeabilized skeletal muscle fibers to assess submaximal and maximal oxidative phosphorylation (OXPHOS; P) primarily linked to complex‐I (CIP), fatty acid oxidation (CIP&amp;FAO), and complex II (CI+IIP&amp;FAO). Subsequent addition of FCCP determined maximal uncoupled electron transport system (ETS) capacity. During the last four weeks of the intervention, participants consumed the stable isotope deuterium oxide (2H2O) to measure cumulative FSR (%/day) in a mitochondrial enriched skeletal muscle fraction. Compared to placebo, Metformin tended (P=0.10) to attenuate the increase in VO2max after 12 weeks of AET (Metformin: 0.06±0.02 vs. Placebo: 0.13±0.03 L/min). Congruent with these findings, Metformin abrogated (P&lt;0.05) the exercise‐mediated increase in submaximal mitochondrial respiration, CIp, CI+IIP, and CI+IIP&amp;FAO after AET (Metformin: −0.7 to 2.6 vs Placebo: 5.3 to 22.8 pmol/s/mg tissue). Unlike the effect of Metformin on mitochondrial respiration, mitochondrial protein synthesis rates assessed during AET were not different between Metformin and placebo (1.06±0.05 vs. 1.00±0.04 %/day; P=0.38). Here we demonstrate that metformin tended to diminish the improvement in VO2max and inhibited the increase in skeletal muscle mitochondrial respiration after AET in older adults at risk for T2D. However, the effects on mitochondrial respiration do not appear to be related to changes in mitochondrial biogenesis. Future research is warranted to determine mechanisms by which metformin influences skeletal muscle mitochondrial respiration and impacts the health benefits of exercise.Support or Funding InformationThis study was supported by the National Dairy CouncilThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Measurement of Muscle, Tendon, Ligament, Cartilage, and Bone Protein Synthesis Rates In Vivo in Humans

IntroductionSkeletal muscle plasticity is reflected by a dynamic balance between protein synthesis and breakdown, with basal muscle protein synthesis rates typically ranging between 0.04 and 0.06 %/h. Though it is evident that other musculoskeletal tissues should also express some level of plasticity, protein synthesis rates of most of these tissues have never been assessed. The present study applies contemporary stable isotope methodology to assess basal muscle, tendon, ligament, bone, as well as cartilage protein synthesis rates in vivo in humans.MethodsSix otherwise healthy patients (62±3 y), scheduled to undergo unilateral total knee arthroplasty, were included in this study. Primed continuous intravenous infusions with L‐[ring‐13C6]‐Phenylalanine were initiated 2.5 h prior to surgery and continued during surgery. Throughout the surgical procedure the following tissue samples were obtained: muscle, tendon, cruciate ligaments, cartilage, bone, menisci, fat and synovium. 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 with muscle tissue protein synthesis rates using a paired t test.ResultsProtein bound L‐[ring‐13C6]‐Phenylalanine enrichments did not differ substantially between the different musculoskeletal tissues and skeletal muscle, except for notch bone tissue (P&lt;0.01). Highest enrichment levels were observed in synovium derived protein (0.029±0.008 MPE) and lowest enrichment levels in patellar bone tissue protein (0.005±0.002 MPE). Tendon, bone, cartilage, fat, anterior cruciate ligament, 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&gt;0.05). Synovium derived protein and notch bone tissue protein synthesis rates were respectively higher and lower compared to skeletal muscle protein synthesis rates (P&lt;0.05 and P&lt;0.01, respectively).ConclusionBasal 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 and menisci tissue protein synthesis rates ranging between 0.02 and 0.13 % per hour.Support or Funding InformationThis study received no specific grant from any funding agency.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Accounting for non‐steady state conditions during long‐term deuterium oxide‐based measurement of protein turnover

IntroductionMost isotope calculations to measure synthesis rates require steady state conditions. In skeletal muscle, steady state refers to a period of time in which there is neither loss or gain of protein. Deuterium oxide (D2O) is a stable isotopic tracer that can measure protein synthesis over long periods of time. This is advantageous when measuring changes in protein synthesis in response to long‐term interventions such as exercise training or pharmacologic interventions. However, prolonging the period of measurement increases the likelihood of non‐steady state conditions. The purpose of this study was to demonstrate that non‐steady state conditions such as growth can change interpretation of tracer data and validate a model that accounts for non‐steady state conditions when assessing muscle protein synthesis. We hypothesized that use of the appropriate model can account for non‐steady state conditions when measuring muscle protein synthesis.MethodsTo demonstrate the potential implications of non‐steady state conditions, we used D2O to measure skeletal muscle protein synthesis over two weeks in three month old male Sprague Dawley rats with access to running wheels during a period of growth. Then we established a model to account for non‐steady state conditions and validated the model in cultured C2C12 myoblasts and myotubes during periods of cell growth and atrophy. Finally, we applied this model in vivo by measuring protein synthesis in four month old male CB6F1 mice with steady weight or weight loss over a month.ResultsDuring a period of growth, active rats gained less body mass than their sedentary counterparts. There was a significant (p&lt;0.05) positive relation between changes in body size and fractional synthesis rates (FSR) (R2 = 0.50, 0.475, 0.50 for myofibrillar, cytosolic, and mitochondrial FSR, respectively) that obscured any exercise‐related increases in protein synthesis. In vitro studies validated the model parameters that rely on a change in protein mass. We grouped mice based on weight maintenance versus weight loss during the period of measurement. We used our modeling parameters to estimate changes in protein synthesis during the steady and non‐steady conditions.ConclusionsNon‐steady state conditions occur with greater likelihood in long‐ versus short‐term protein synthesis studies and should be accounted for. We showed that a period of growth could account for ~50% of all variation in changes in protein synthesis in all subfractions of skeletal muscle, and this growth effect could mask more subtle effects that may be important for hypothesis testing. We have developed a model to account for non‐steady state conditions over long‐term interventions. We applied this model to a one month intervention to account for body mass changes in response to dietary restriction. By applying this model to future long‐term interventions, we can more accurately discern the effects of interventions on protein synthesis in non steady‐state conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal Muscle Disuse Alters Exosome miRNA Predicted to Target Various Signaling Pathways Related to Muscle Atrophy

Skeletal muscle atrophy that occurs during disuse is linked to poor functional and metabolic health outcomes. Small noncoding microRNAs (miRNAs) are now appreciated as important components in the regulation of muscle growth and atrophy and can be carried through systemic circulation in exosomes to potentially mediate systemic adaptations. However, little is known about how atrophic conditions influence the miRNA content of exosomes. The purpose of this study was to determine changes in exosome miRNA in response to atrophic conditions. Adult (10 months old) Brown Norway/F344 male rats were subjected to either normal, weight‐bearing conditions (WB: n=7) or a 7‐day hindlimb suspension (HS: n=6) protocol that is known to induce significant skeletal muscle atrophy in select hindlimb muscles. Rats received deuterium oxide (D2O) over 10 days to label newly synthesized proteins for determination of fractional synthesis rate (FSR, %/day). Rat serum was collected and filtered through a 0.22μm filter for removal of larger extracellular vesicles. Exosomes were isolated using the ExoQuick™ Precipitation solution before isolation of the exosome miRNA. As expected, HS had lower myofibrillar skeletal muscle FSR compared with WB rats (0.72 ± 0.09%/day vs 1.64 ± 0.27%/day; p&lt;0.05). MiRNA microarray analysis of exosome miRNA revealed 73 differentially expressed (p&lt;0.05) miRNA between WB and HS conditions. The list of differentially expressed exosome miRNAs was uploaded to DIANA‐miRPath for pathway analysis and prediction of regulatory pathways potentially impacted by the altered exosome miRNAs during atrophy. Various pathways related to muscle protein homeostasis were significantly predicted (p&lt;0.01) to be targeted by the differentially expressed exosome miRNA, including MAPK signaling, focal adhesion, Rap1 signaling, FOXO signaling, ubiquitin mediate proteolysis, TGF‐beta, and mTOR signaling. These data suggest that alterations in systemic exosome miRNA reflect changes in the major pathways dictating muscle atrophy during muscle disuse. In addition, selective packaging and release of exosomes containing miRNA important for regulation of muscle atrophy may be a major contributor to determining skeletal muscle homeostasis during muscle disuse.Support or Funding InformationNIH grants AT009268 and AG042699This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipoprotein turnover and possible remnant accumulation in preeclampsia: insights from the Freiburg Preeclampsia H.E.L.P.-apheresis study

BackgroundPreeclampsia is a life-threatening disease in pregnancy, and its complex pathomechanisms are poorly understood. In preeclampsia, lipid metabolism is substantially altered. In late onset preeclampsia, remnant removal disease like lipoprotein profiles have been observed. Lipid apheresis is currently being explored as a possible therapeutic approach to prolong preeclamptic pregnancies. Here, apheresis-induced changes in serum lipid parameters are analyzed in detail and their implications for preeclamptic lipid metabolism are discussed.MethodsIn the Freiburg H.E.L.P.-Apheresis Study, 6 early onset preeclamptic patients underwent repeated apheresis treatments. Serum lipids pre- and post-apheresis and during lipid rebound were analyzed in depth via ultracentrifugation to yield lipoprotein subclasses.ResultsThe net elimination of Apolipoprotein B and plasma lipids was lower than theoretically expected. Lipids returned to previous pre-apheresis levels before the next apheresis even though apheresis was repeated within 2.9 ± 1.2 days. Apparent fractional catabolic rates and synthetic rates were substantially elevated, with fractional catabolic rates for Apolipoprotein B / LDL-cholesterol being 0.7 ± 0.3 / 0.4 ± 0.2 [day− 1] and synthetic rates being 26 ± 8 / 17 ± 8 [mg*kg− 1*day− 1]. The distribution of LDL-subclasses after apheresis shifted to larger buoyant LDL, while intermediate-density lipoprotein-levels remained unaffected, supporting the notion of an underlying remnant removal disorder in preeclampsia.ConclusionLipid metabolism seems to be highly accelerated in preeclampsia, likely outbalancing remnant removal mechanisms. Since cholesterol-rich lipoprotein remnants are able to accumulate in the vessel wall, remnant lipoproteins may contribute to the severe endothelial dysfunction observed in preeclampsia.Trial registrationClinicalTrails.gov, NCT01967355.

Propranolol and Oxandrolone Therapy Accelerated Muscle Recovery in Burned Children.

Severe burns result in prolonged hypermetabolism and skeletal muscle catabolism. Rehabilitative exercise training (RET) programs improved muscle mass and strength in severely burned children. The combination of RET with β-blockade or testosterone analogs showed improved exercise-induced benefits on body composition and muscle function. However, the effect of RET combined with multiple drug therapy on muscle mass, strength, cardiorespiratory fitness, and protein turnover are unknown. In this placebo-controlled randomized trial, we hypothesize that RET combined with oxandrolone and propranolol (Oxprop) will improve muscle mass and function and protein turnover in severely burned children compared with burned children undergoing the same RET with a placebo. We studied 42 severely burned children (7-17 yr) with severe burns over 30% of the total body surface area. Patients were randomized to placebo (22 control) or to Oxprop (20) and began drug administration within 96 h of admission. All patients began RET at hospital discharge as part of their standardized care. Muscle strength (N·m), power (W), V˙O2peak, body composition, and protein fractional synthetic rate and fractional breakdown rate were measured pre-RET (PRE) and post-RET (POST). Muscle strength and power, lean body mass, and V˙O2peak increased with RET in both groups (P < 0.01). The increase in strength and power was significantly greater in Oxprop versus control (P < 0.01), and strength and power was greater in Oxprop over control POST (P < 0.05). Fractional synthetic rate was significantly higher in Oxprop than control POST (P < 0.01), resulting in improved protein net balance POST (P < 0.05). Rehabilitative exercise training improves body composition, muscle function, and cardiorespiratory fitness in children recovering from severe burns. Oxprop therapy augments RET-mediated improvements in muscle strength, power, and protein turnover.

Even effect of milk protein and carbohydrate intake but no further effect of heavy resistance exercise on myofibrillar protein synthesis in older men.

The responsiveness of older individuals' skeletal muscle to anabolic strategies may be impaired. However, direct comparisons within the same experimental setting are sparse. The aim of this study was to assess the resting and post-resistance exercise muscle protein synthesis rates in response to two types of milk protein and carbohydrate using a unilateral exercise leg model. Twenty-seven older (69 ± 1year, mean ± SE) men were randomly assigned one of three groups: Whey hydrolysate (WH), caseinate (CAS), or carbohydrate (CHO). By applying stable isotope tracer techniques (L-[15N]phenylalanine), the fasted-rested (basal) myofibrillar fractional synthesis rate (FSR) was measured. Hereafter, FSR was measured in the postprandial phase (0.45g nutrient/kg LBM) in both legs, one rested (fed-rest) and one exercised (10 × 8 reps at 70% 1RM; fed-exercise). In addition, the activity of p70S6K and venous plasma insulin, phenylalanine, and leucine concentrations were measured. Insulin, phenylalanine, and leucine concentrations differed markedly after intake of the different study drinks. The basal FSR in WH, CAS, and CHO were 0.027 ± 0.003, 0.030 ± 0.003, and 0.030 ± 0.004%/h, the fed-rested FSR were 0.043 ± 0.004, 0.045 ± 0.003, and 0.035 ± 0.004%/h, and the fed-exercised FSR were 0.041 ± 0.004, 0.043 ± 0.004, and 0.034 ± 0.004%/h, respectively. No significant differences were observed at any state between the groups. Fed-rested- and fed-exercised FSR were higher than basal (P < 0.001). 3h after exercise and feeding, no significant group differences were detected in the activity of p70S6K. Milk protein and carbohydrate supplementation stimulate myofibrillar protein synthesis in older men, with no further effect of heavy resistance exercise within 0-3h post exercise.

Impact of 3-week citrulline supplementation on postprandial protein metabolism in malnourished older patients: The Ciproage randomized controlled trial

Citrulline (CIT), is not extracted by the splanchnic area, can stimulate muscle protein synthesis and could potentially find clinical applications in conditions involving low amino acid (AA) intake, such as in malnourished older subjects. Our purpose was to research the effects of CIT supplementation on protein metabolism in particular on non-oxidative leucine disposal (NOLD, primary endpoint), and splanchnic extraction of amino acids in malnourished older patients. This prospective randomized multicenter study determined whole-body and liver protein synthesis, splanchnic protein metabolism and appendicular skeletal muscle mass (ASMM) in 24 malnourished older patients [80-92 years; 18 women and 6 men] in inpatient rehabilitation units. All received an oral dose of 10g of CIT or an equimolar mixture of six non-essential amino acids (NEAAs), as isonitrogenous placebo, for 3 weeks. NOLD and albumin fractional synthesis rates were not different between the NEAA and CIT groups. Splanchnic extraction of dietary amino acid tended to decrease (p=0.09) in the CIT group (45.2%) compared with the NEAA group (60.3%). Total differences in AA and NEAA area under the curves between fed-state and postabsorptive-state were significantly higher in the CIT than in the NEAA group. There were no significant differences for body mass index, fat mass (FM), lean mass (LM) or ASMM in the whole population except for a tendential decrease in FM for the citrulline group (p=0.089). Compared with Day 1, lean mass and ASMM significantly increased (respectively p=0.016 and p=0.018) at Day 20 in CIT-treated women (mean respective increase of 1.7kg and 1.1kg), and fat mass significantly decreased (p=0.001) at Day 20 in CIT-group women (mean decrease of 1.3kg). Our results demonstrate that CIT supplementation has no effect on whole-body protein synthesis or liver protein synthesis in malnourished older subjects. However, CIT supplementation was associated with a higher systemic AA availability. In the subgroup of women, CIT supplementation increased LM and ASMM, and decreased FM.

The effect of sexual maturity and egg production on skeletal muscle (pectoralis major and gastrocnemius) protein turnover in broiler breeder pure lines

A study was conducted to evaluate the effect of sexual maturity on pectoralis major and gastrocnemius muscle protein turnover in broiler breeder pure lines. Protein turnover in skeletal muscle tissue was determined in 4 broiler breeder pure lines (Line A, Line B, Line C and Line D) at 22, 27, 33, 37, 44, and 50 wk of age. A completely randomized design with a factorial arrangement 4 × 6 (4 lines and 6 time periods (ages)) was used. There were 5 replicates per line/time and each hen represented a replicate. Five hens/line at each age were given an intravenous flooding-dose of 15 N-phenylalanine (150 mM, 40 atom percent excess (APE) at a dose of 10 mL/kg. After 10 min, birds were euthanized using CO2 asphyxiation and the breast and leg muscle excised and snap frozen in liquid nitrogen for protein turnover analysis. Excreta was collected from each breeder for 3-methyl histidine (3-MH) analysis. There was a significant age effect for the breast muscle fractional synthesis rate (FSR), but no main effects (age and line) for leg muscle FSR. The FSR in breast muscle tissue decreased in hens from wk 22 (first egg) to wk 33 (peak egg production). There was a significant age effect on fractional breakdown rate (FBR) in breast and leg muscle. The FBR in breast muscle increased in hens from wk 22 to wk 33 and remained high through wk 37. Breast muscle FBR significantly decreased in hens from wk 37 to wk 50. The FBR in leg muscle tissue increased in hens from wk 33 to wk 37 and then decreased at wk 50. No line effect was seen for FSR or FBR. There is a large increase in skeletal muscle FBR during the transition for the pullet to sexual maturity with increases in skeletal muscle FBR in the breast and leg muscle through peak egg production. Protein turnover in skeletal muscle tissue is believed to be a source of nutrients for egg production.

Sulfur amino acid restriction-induced changes in redox-sensitive proteins are associated with slow protein synthesis rates.

The mechanisms underlying life span extension by sulfur amino acid restriction (SAAR) are unclear. Cysteine and methionine are essential for the biosynthesis of proteins and glutathione (GSH), a major redox buffer in the endoplasmic reticulum (ER). We hypothesized that SAAR alters protein synthesis by modulating the redox milieu. Male F344-rats were fed control (CD: 0.86% methionine without cysteine) and SAAR diets (0.17% methionine without cysteine) for 12 weeks. Growth rates, food intake, cysteine and GSH levels, proteins associated with redox status and translation, and fractional protein synthesis rates (FSRs) were determined in liver. Despite a 40% higher food intake, growth rates for SAAR rats were 27% of those fed CD. Hepatic free cysteine in SAAR rats was 55% compared with CD rats. SAAR altered tissue distribution of GSH, as hepatic and erythrocytic levels were 56% and 196% of those in CD rats. Lower GSH levels did not induce ER stress (i.e., unchanged expression of Xbp1s , Chop, and Grp78), but activated PERK and its substrates eIF2-α and NRF2. SAAR-induced changes in translation-initiation machinery (higher p-eIF2-α and 4E-BP1, and lower eIF4G-1) resulted in slower protein synthesis rates (53% of CD). Proteins involved in the antioxidant response (NRF2, KEAP1, GCLM, and NQO1) and protein folding (PDI and ERO1-α) were increased in SAAR. Lower FSR and efficient protein folding might be improving proteostasis in SAAR.

Enhanced skeletal muscle regrowth and remodelling in massaged and contralateral non-massaged hindlimb.

Muscle fibre cross sectional area is enhanced with massage in the form of cyclic compressive loading during regrowth after atrophy. Massage enhances protein synthesis of the myofibrillar and cytosolic, but not the mitochondrial fraction, in muscle during regrowth. Focal adhesion kinase activation and satellite cell number are elevated in muscles undergoing massage during regrowth. Muscle fibre cross sectional area and protein synthesis of the myofibrillar fraction, but not DNA synthesis, are elevated in muscle of the contralateral non-massaged limb. Massage in the form of cyclic compressive loading is a potential anabolic intervention during muscle regrowth after atrophy. Massage, in the form of cyclic compressive loading (CCL), is associated with multiple health benefits, but its potential anabolic effect on atrophied muscle has not been investigated. We hypothesized that the mechanical activity associated with CCL induces an anabolic effect in skeletal muscle undergoing regrowth after a period of atrophy. Fischer-Brown Norway rats at 10months of age were hindlimb unloaded for a period of 2weeks. The rats were then allowed reambulation with CCL applied at a 4.5N load at 0.5Hz frequency for 30min every other day for four bouts during a regrowth period of 8days. Muscle fibre cross sectional area was enhanced by 18% with massage during regrowth compared to reloading alone, and this was accompanied by elevated myofibrillar and cytosolic protein as well as DNA synthesis. Focal adhesion kinase phosphorylation indicated that CCL increased mechanical stimulation, while a higher number of Pax7+ cells likely explains the elevated DNA synthesis. Surprisingly, the contralateral non-massaged limb exhibited a comparable 17% higher muscle fibre size compared to reloading alone, and myofibrillar protein synthesis, but not DNA synthesis, was also elevated. We conclude that massage in the form of CCL induces an anabolic response in muscles regrowing after an atrophy-inducing event. We suggest that massage can be used as an intervention to aid in the regrowth of muscle lost during immobilization.