Basal Protein Synthesis Rates Research Articles

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

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

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

USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signaling in muscle during prolonged starvation.

PI3K-Akt-FoxO-mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K-Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K-Akt-FoxO signaling, and glucose uptake during fasting. Co-immunoprecipitation and mass spectrometry identified disabled homolog-2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1-TSC1-PHLPP1 complex, and for PI3K-Akt-FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1-TSC1-PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low.

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

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.

Ribosomal Capacity’s Relationship To Muscle Oxidative Metabolism

Skeletal muscle’s capacity for oxidative metabolism parallels the basal rate of protein synthesis and ribosomal capacity. Muscle metabolic activity and protein synthesis are regulated by both muscle contraction and cytokine signaling. While endurance exercise induces oxidative metabolism, a role for ribosomal capacity in these changes is not well understood. The interleukin-6 (IL-6) cytokine family through the glycoprotein 130 receptor (gp130) induces cellular signaling that regulate muscle metabolism and remodeling. While muscle oxidative metabolism is induced by exercise, the regulatory role of ribosomal capacity and the IL-6 cytokine family for this induction is not known PURPOSE: To examine the relationship between muscle oxidative metabolism and ribosomal capacity in basal and trained muscle, and determine the potential regulation by gp130 signaling. METHODS: Male C57BL/6 (B6; N=18) and skeletal muscle specific gp130 knockout (KO; N=17) mice were randomly selected to either cage control or treadmill exercise. Treadmill training (6 day/wk., 1 h/d) was initiated at 6 weeks of age and mice were sacrificed at 12 wks. of age. Quadriceps muscle cytochrome c oxidase (COX) enzyme activity, and total RNA and protein were examined. RESULTS: At baseline B6 COX activity was positively correlated with total RNA content (R2=0.63, p=0.01), but not in KO muscle (R2=0.02, p=0.65). Exercise increased B6 and KO COX activity (p<0.0001), and ablated the relationship between COX activity and total RNA content in B6 muscle (R2=0.01, p=0.90). CONCLUSIONS: These data demonstrate that basal COX enzyme activity is positively associated with ribosomal capacity, but increased ribosomal capacity is not required for the exercise induction of COX activity. Additionally, the relationship between basal muscle oxidative metabolism and ribosomal capacity requires muscle gp130 signaling. Supported by NCI R01-CA121249

Nutritionally essential amino acids and metabolic signaling in aging

Aging is associated with a gradual decline in skeletal muscle mass and strength leading to increased risk for functional impairments. Although basal rates of protein synthesis and degradation are largely unaffected with age, the sensitivity of older muscle cells to the anabolic actions of essential amino acids appears to decline. The major pathway through which essential amino acids induce anabolic responses involves the mammalian target of rapamycin (mTOR) Complex 1, a signaling pathway that is especially sensitive to regulation by the branched chain amino acid leucine. Recent evidence suggests that muscle of older individuals require increasing concentrations of leucine to maintain robust anabolic responses through the mTOR pathway. While the exact mechanisms for the age-related alterations in nutritional signaling through the mTOR pathway remain elusive, there is increasing evidence that decreased sensitivity to insulin action, reductions in endothelial function, and increased oxidative stress may be underlying factors in this decrease in anabolic sensitivity. Ensuring adequate nutrition, including sources of high quality protein, and promoting regular physical activity will remain among the frontline defenses against the onset of sarcopenia in older individuals.

Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

β2 Adrenoceptor Signaling-induced Muscle Hypertrophy from Blood Flow Restriction: Is There Evidence?

Skeletal muscle hypertrophy and increases in muscular function have been observed following low intensity/load exercise with blood flow restriction (BFR). The mechanisms behind these effects are largely unknown, but have been hypothesized to include a metabolic accumulation induced increase in muscle activation, elevations in growth hormone, and improvements in muscle protein balance. However, many of the aforementioned mechanisms are not present with BFR in the absence of exercise. In these situations, signaling through the β2 adrenoceptor has been hypothesized to possibly contribute to the positive muscle adaptions, possibly in concert with muscle cell swelling. Signaling through the β2 adrenoceptor has been shown to stimulate both muscle protein synthesis and an inhibition of protein degradation through increasing cyclic adenosine monophosphate (cAMP) or signaling via the Gβγ subunit, especially in situations where the basal rates of protein synthesis are already reduced. Every study that has investigated the catecholamine response to BFR in the absence of exercise or in combination with exercise has shown a significant increase above resting conditions. However, from the available evidence, it is unlikely that the norepinephrine response from BFR, particularly with exercise, is playing a prominent role with muscle adaptation in skeletal muscle that is not immobilized by a cast or joint injury.

Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Selective vulnerability of neurons to acute toxicity after proteasome inhibitor treatment: Implications for oxidative stress and insolubility of newly synthesized proteins

Maintaining protein homeostasis is vital to cell viability, with numerous studies demonstrating a role for proteasome inhibition occurring during the aging of a variety of tissues and, presumably, contributing to the disruption of cellular homeostasis during aging. In this study we sought to elucidate the differences between neurons and astrocytes in regard to basal levels of protein synthesis, proteasome-mediated protein degradation, and sensitivity to cytotoxicity after proteasome inhibitor treatment. In these studies we demonstrate that neurons have an increased vulnerability, compared to astrocyte cultures, to proteasome-inhibitor-induced cytotoxicity. No significant difference was observed between these two cell types in regard to the basal rates of protein synthesis, or basal rates of protein degradation, in the pool of short-lived proteins. After proteasome inhibitor treatment neuronal crude lysates were observed to undergo greater increases in the levels of ubiquitinated and oxidized proteins and selectively exhibited increased levels of newly synthesized proteins accumulating within the insoluble protein pool, compared to astrocytes. Together, these data suggest a role for increased oxidized proteins and sequestration of newly synthesized proteins in the insoluble protein pool, as potential mediators of the selective neurotoxicity after proteasome inhibitor treatment. The implications for neurons exhibiting increased sensitivity to acute proteasome inhibitor exposure, and the corresponding changes in protein homeostasis observed after proteasome inhibition, are discussed in the context of both aging and age-related disorders of the nervous system.

A Method To Study In Vivo Protein Synthesis In Slow- And Fast-twitch Human Muscle Fibers

PURPOSE: To develop an approach to directly assess protein fractional synthesis rate (FSR) in isolated human muscle fibers and to examine potential differences in FSR between slow (MHC I) and fast twitch (MHC IIa) fibers. METHODS: Individual muscle fibers were isolated from biopsies of the vastus lateralis (VL) and soleus (SOL) obtained from eight young men during a primed, continuous infusion of 2H3 leucine performed under basal conditions. To determine mixed protein FSR, a portion of each fiber was used to identify fiber type, fibers of the same fiber type were pooled, and the 2H3 leucine enrichment determined via GC-MS. RESULTS: In the VL, MHC I fibers exhibited a 33% faster (p<0.05) mixed protein FSR compared to MHC IIa fibers (0.0900.005 vs. 0.0680.002 %/hr). Similar (p>0.05) mixed protein FSR values were observed between MHC I fibers from the SOL (0.0820.006 %/hr) and MHC I fibers from the VL. Feasibility of processing isolated fibers for myofibrillar (MF) protein FSR was also determined in non-fiber typed pooled fibers from the VL, resulting in a MF protein FSR of 0.0730.004 %/hr. CONCLUSION: These methods can be applied to the study of fiber type specific responses in human skeletal muscle, and the need for this level of investigation is underscored by the different basal protein synthesis rates in MHC I and MHC IIa fibers originating from the same muscle. Supported by NIH R01 AG025032; NASA NNJ06HF59G

Treatment with IFN‐γ prevents insulin‐dependent PKB, p70S6k phosphorylation and protein synthesis in mouse C2C12 myogenic cells

The purpose of the present study was to examine the potential effect of IFN-gamma (interferon-gamma) on the cellular content and phosphorylation of PKB (protein kinase B), p70S6k (p70 S6 kinase) and MAPK (mitogen-activated protein kinase), and on the ability of insulin to stimulate the glucose uptake and protein synthesis in mouse C2C12 myotubes. Insulin (100 nmol/l) stimulated glucose uptake in C2C12 myotubes by 203.4%. Glucose uptake in cells differentiated in the presence of IFN-gamma (10 ng/ml) was increased by 165.8% and was not further significantly modified by the addition of insulin (183.4% of control value). Insulin increased the rate of protein synthesis by 198.8%. The basal rate of protein synthesis was not affected by IFN-gamma; however, this cytokine abolished the insulin effect. Cellular levels of PKB, p70S6k, p42MAPK and p44MAPK were not modified by IFN-gamma. Insulin caused the phosphorylation of PKB and the activation of p70S6k, but not p42MAPK and p44MAPK. In cells differentiated in the presence of IFN-gamma, the insulin-mediated PKB phosphorylation was significantly diminished, whereas the phosphorylation of p70S6k was completely prevented. Pretreatment of C2C12 myogenic cells with IFN-gamma led to the marked increase in p42MAPK phosphorylation. Exposure of C2C12 myoblasts to IFN-gamma impaired MyoD and myogenin expression and decreased the fusion index on the fifth day of differentiation. In conclusion, (i) IFN-gamma present in the extracellular environment during C2C12 myoblast differentiation prevents the stimulatory action of insulin on protein synthesis; (ii) IFN-gamma-induced insulin resistance of protein synthesis in myogenic cells can be associated with the decreased phosphorylation of PKB and p70S6k, as well as with the augmented basal phosphorylation of p42MAPK; (iii) this cytokine effect can be partly explained by alterations in the differentiation process.

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin‐induced increase in global rates of muscle protein synthesis during neonatal sepsis involves an increase in protein synthesis in the mitochondria, neonatal pigs (n=16) underwent cecal ligation and puncture or sham surgery and were pair‐gavage fed for 5 days. Pancreatic‐substrate clamps were performed to achieve fasting or refed insulin levels and fractional protein synthesis rates and translational control mechanisms were examined in muscle. CLP increased IL‐8 and CRP, but not TNF‐α, and reduced weight gain. In fasted pigs, CLP reduced global and mitochondrial fractional protein synthesis in muscle. Raising insulin increased global and mitochondrial fractional muscle protein synthesis rates in sham and CLP animals, but increased eIF4G‐eIF4E association in shams only. The results suggest that mitochondrial dysfunction contributes to the sepsis‐associated decrease in basal protein synthesis rates in skeletal muscle, which may be ameliorated by insulin stimulation. NIH AR44474, NIH AR51563, NIH HD41648, and USDA 58‐6250‐6‐001.

Endotoxin Reduces Muscle Protein Synthesis and Restrains Translation Initiation by Decreasing eIF4G Phosphorylation in Neonatal and Young Pigs

Endotoxin (LPS) reduces muscle protein synthesis by restraining translation in the presence of fed levels of insulin and amino acids. In vivo effects of LPS on translation initiation in muscle during basal fasting insulin and amino acid conditions have not been determined in neonates, whose muscle protein synthesis rates are uniquely high. Overnight fasted 7‐ and 26‐d‐old pigs were infused for 8 h with LPS (0 and 10 μg·kg−1·hr−1) and fractional protein synthesis rates and translational control mechanisms in skeletal muscle were examined (n=5–7/group/age). LPS reduced the basal rate of muscle protein synthesis in 7‐ and 26‐day‐old pigs in the presence of fasting levels of insulin, glucose, and amino acids. LPS decreased eIF4G phosphorylation and the amount of the active eIF4G‐eIF4E complex in muscle of 7‐ and 26‐d‐old pigs, but had no effect on mTOR or eEF2 phosphorylation. Although muscle protein synthesis decreased and eEF2 phosphorylation increased with age, there was no effect of age on translation initiation factor activation in basal fasting conditions. The results suggest that the LPS‐induced decrease in eIF4G phosphorylation in muscle may be a limiting step in translation initiation which contributes to the sepsis‐associated decrease in basal protein synthesis rates in muscle during early postnatal life. NIH AR44474, NIH AR51563, NIH HD41648, and USDA 58‐6250‐6‐001.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Abstracts for Poster Presentation

Abstracts for Poster Presentation

HIV Antiretroviral Agents Inhibit Protein Synthesis and Decrease Ribosomal Protein S6 and 4EBP1 Phosphorylation in C2C12 Myocytes

Combined antiretroviral drug regimens have promoted clinical, immunologic, and virologic improvements in AIDS patients. Nevertheless, these therapies are associated with derangements in lipid and carbohydrate metabolism. In this study, we examined the effects of a representative protease inhibitor (nelfinavir), a nonnucleoside reverse transcriptase inhibitor (nevirapine), and a nucleoside reverse transcriptase inhibitor (zidovudine) on protein synthesis in skeletal muscle cells. To examine these processes, C2C12 myocytes were treated with increasing concentrations of nelfinavir, nevirapine, or zidovudine for 1 or 2 days, and rates of protein synthesis were determined by measuring [35S]methionine/cysteine incorporation into cellular proteins. Treatment of myocytes with therapeutic concentrations of nelfinavir, nevirapine, or zidovudine for 48 hr decreased protein synthesis by 14-20%. An approximately 60% decline was observed in cells treated with higher concentrations of nevirapine or nelfinavir. In contrast, the basal rate of protein synthesis was not affected when cells were incubated with these compounds for 24 hr. Therapeutic concentrations of nelfinavir and nevirapine did not impair the anabolic effect of insulin on protein synthesis. However, zidovudine suppressed the stimulatory effect of insulin. The decreased protein synthesis induced by nelfinavir and zidovudine was associated with decreases in the phosphorylation of the S6 ribosomal protein (rpS6) and the repressor binding protein 4EBP1, while the inhibitory effect of nevirapine was mainly associated with a decline in phosphorylated 4EBP1. In conclusion, nelfinavir, nevirapine, and zidovudine treatments decreased protein synthesis in myocytes and this effect was correlated with a reduction in the phosphorylation level of proteins that regulate translation initiation.