Protein Synthesis In Response Research Articles

Anabolic Heterogeneity Following Resistance Training: A Role for Circadian Rhythm?

It is now well established that resistance exercise stimulates muscle protein synthesis and promotes gains in muscle mass and strength. However, considerable variability exists following standardized resistance training programs in the magnitude of muscle cross-sectional area and strength responses from one individual to another. Several studies have recently posited that alterations in satellite cell population, myogenic gene expression and microRNAs may contribute to individual variability in anabolic adaptation. One emerging factor that may also explain the variability in responses to resistance exercise is circadian rhythms and underlying molecular clock signals. The molecular clock is found in most cells within the body, including skeletal muscle, and principally functions to optimize the timing of specific cellular events around a 24 h cycle. Accumulating evidence investigating the skeletal muscle molecular clock indicates that exercise-induced contraction and its timing may regulate gene expression and protein synthesis responses which, over time, can influence and modulate key physiological responses such as muscle hypertrophy and increased strength. Therefore, the circadian clock may play a key role in the heterogeneous anabolic responses with resistance exercise. The central aim of this Hypothesis and Theory is to discuss and propose the potential interplay between the circadian molecular clock and established molecular mechanisms mediating muscle anabolic responses with resistance training. This article begins with a current review of the mechanisms associated with the heterogeneity in muscle anabolism with resistance training before introducing the molecular pathways regulating circadian function in skeletal muscle. Recent work showing members of the core molecular clock system can regulate myogenic and translational signaling pathways is also discussed, forming the basis for a possible role of the circadian clock in the variable anabolic responses with resistance exercise.

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<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.

Fine‐tuning of hepatocyte calcium signaling and liver regeneration by the mitochondrial calcium uniporter

Calcium (Ca2+) signaling regulates a variety of cellular processes such as metabolism, cell proliferation, division and differentiation, gene transcription, stimulus‐secretion coupling and cell death. Ca2+ is able to regulate these diverse cellular processes due to the versatility of the Ca2+ signaling mechanism in terms of speed, amplitude and spatiotemporal patterning. Mitochondria form an integral part of this machinery, both as regulators and targets of Ca2+ signaling. In recent years, leading‐edge research in the field of mCa2+ (mitochondrial Ca2+) handling and its implications for cellular Ca2+ homeostasis opened up the possibility to investigate the functional implications of mCa2+ uptake in shaping the spatiotemporal dynamics of Ca2+ signaling, and regulating cellular metabolism (through activation of Ca2+ sensitive TCA cycle enzymes) and mitochondria‐mediated cell death (through mitochondrial permeability transition). mCa2+ is mediated by the mitochondrial uniporter complex, consisting of the pore forming MCU (Mitochondrial Calcium Uniporter) and regulators such as MICU1 (mitochondrial calcium uptake 1), MICU2 and EMRE (essential MCU regulator). MCU deletion abolishes mCa2+ uptake in mouse hepatocytes driven by vasopressin‐evoked cCa2+ (cytosolic Ca2+) increases. This lack of mCa2+ uptake resulted in alterations in cCa2+ dynamics, including an elevated resting [cCa2+] and a reduced cCa2+ peak and the total Ca2+ mobilization in response to vasopressin stimulation. These changes in cCa2+ dynamics could lead to alterations in downstream Ca2+ responsive signaling events. Indeed, we see suppression of AMPK activation through CAMKK2, in response to hormone‐evoked Ca2+ signals in MCU KD hepatocytes. Activation of Creb, CAMKK2, AMPKα and ERK1/2 are also more transient in the MCU KD hepatocytes compared to controls. Our previous studies have highlighted the importance of regulating MCU (through MICU1) under stress conditions involving enhanced mCa2+ entry, especially during liver regeneration following partial hepatectomy (PHx). Enhanced mCa2+ entry during liver regeneration maybe vital to cellular signaling events and increased mitochondrial ATP production. The lack of mCa2+ uptake in hepatocyte‐specific MCU KD mice resulted in decelerated kinetics of cell cycle progression after PHx reflected by the inhibition in BrdU incorporation and Ki67 upregulation at 30h post‐PHx, which recovers by 36h. Interestingly, apart from an inhibition in Cyclin D1 expression, many cell cycle regulatory proteins remained unchanged in the KD compared to WT controls. Cell signaling events such as AMPK, CAMKK2 and mTOR activation post‐PHx are altered. Gene expression analysis at 30h post‐PHx revealed massive changes in gene expression profiles between KD and control livers, especially in genes involving metabolic processes and protein synthesis in response to PHx. Overall, the lack of mCa2+ uptake affects multiple signaling and metabolic pathways in hepatocytes and impacts the kinetics of the liver regeneration response.Support or Funding InformationAA018873 and U01EB023224 (JBH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Origins of robustness in translational control via eukaryotic translation initiation factor (eIF) 2

Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. This pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness-conferring features. Our analyses indicate that robustness is a distributed property, rather than arising from the properties of any one individual pathway species. However, robustness-conferring properties are unevenly distributed between the different species, and we identify a guanine nucleotide dissociation inhibitor (GDI) complex as a species that likely contributes strongly to the robustness of the pathway. Our analyses make further predictions on the dynamic response to different types of kinases that impinge on eIF2.

Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial

Older women may not be consuming enough protein to maintain muscle mass. Augmentation of protein intake with leucine may enhance the muscle protein synthetic response in older women to aid in maintaining muscle mass. We measured the acute (hourly) and integrated (daily) myofibrillar protein synthesis (myoPS) response to consumption of a high-quality mixed protein beverage compared with an isonitrogenous protein beverage with added leucine. In a parallel design, free-living, healthy older women (aged 65-75 y, n = 11/group) consumed a fixed, weight-maintaining diet with protein at 1.0 g · kg-1 · d-1 and were randomly assigned to twice-daily consumption of either 15 g milk protein beverage containing 4.2 g leucine (LEU) or 15 g mixed protein (milk and soy) beverage containing 1.3 g leucine (CON). Unilateral leg resistance exercise allowed a determination of acute ([13C6]-phenylalanine infusion, hourly rate) and integrated (deuterated water ingestion, daily rate) exercised and rested myoPS responses. Acute myoPS increased in response to feeding in the rested (CON: 13% ± 4%; LEU: 53% ± 5%) and exercised (CON: 30% ± 4%; LEU: 87% ± 7%) leg in both groups, but the increase was greater in LEU (P < 0.001). Integrated myoPS increased during the supplementation period in both legs (rested: 9% ±1%; exercised: 17% ± 2%; P < 0.001) in LEU, but in the exercised leg only (7% ± 2%; P < 0.001) in CON. A 15-g protein-containing beverage with ∼4 g leucine induced greater increases in acute and integrated myoPS than did an isonitrogenous, isoenergetic mixed-protein beverage. Declines in muscle mass in older women may be attenuated with habitual twice-daily consumption of a protein beverage providing 15 g protein and higher (4.2 g/serving) amounts of leucine. This trial was registered at clinicaltrials.gov as NCT02282566.

Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications

Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3′ end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Increased skeletal muscle IL-15Rα is associated with myofibrillar protein synthesis in response to a single session of resistance exercise

Increased skeletal muscle IL-15Rα is associated with myofibrillar protein synthesis in response to a single session of resistance exercise

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Inflammatory signalling regulates eccentric contraction-induced protein synthesis in cachectic skeletal muscle.

BackgroundSkeletal muscle responds to eccentric contractions (ECC) with an anabolic response that involves the induction of protein synthesis through the mechanistic target of rapamycin complex 1. While we have reported that repeated ECC bouts after cachexia initiation attenuated muscle mass loss and inflammatory signalling, cachectic muscle's capacity to induce protein synthesis in response to ECC has not been determined. Therefore, we examined cachectic muscle's ability to induce mechano‐sensitive pathways and protein synthesis in response to an anabolic stimulus involving ECC and determined the role of muscle signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) signalling on ECC‐induced anabolic signalling.MethodsMechano‐sensitive pathways and anabolic signalling were examined immediately post or 3 h after a single ECC bout in cachectic male Apc Min/+ mice (n = 17; 16 ± 1% body weight loss). Muscle STAT3/NFκB regulation of basal and ECC‐induced anabolic signalling was also examined in an additional cohort of Apc Min/+ mice (n = 10; 16 ± 1% body weight loss) that received pyrrolidine dithiocarbamate 24 h prior to a single ECC bout. In all experiments, the left tibialis anterior performed ECC while the right tibialis anterior served as intra‐animal control. Data were analysed by Student's t‐test or two‐way repeated measures analysis of variance with Student‐Newman‐Keuls post‐hoc when appropriate. The accepted level of significance was set at P < 0.05 for all analysis.Results Apc Min/+ mice exhibited a cachectic muscle signature demonstrated by perturbed proteostasis (Ribosomal Protein S6 (RPS6), P70S6K, Atrogin‐1, and Muscle RING‐finger protein‐1 (MuRF1)), metabolic (adenosine monophosphate‐activated protein kinase, Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α), and Cytochrome c oxidase subunit IV (COXIV)), and inflammatory (STAT3, NFκB, extracellular signal‐regulated kinases 1 and 2, and P38) signalling pathway regulation. Nonetheless, mechano‐sensitive signalling pathways (P38, extracellular signal‐regulated kinases 1 and 2, and Protein kinase B (AKT)) were activated immediately post‐ECC irrespective of cachexia. While cachexia did not attenuate ECC‐induced P70S6K activation, the protein synthesis induction remained suppressed compared with healthy controls. However, muscle STAT3/NFκB inhibition increased basal and ECC‐induced protein synthesis in cachectic Apc Min/+ mice.ConclusionsThese studies demonstrate that mechano‐sensitive signalling is maintained in cachectic skeletal muscle, but chronic STAT3/NFκB signalling serves to attenuate basal and ECC‐induced protein synthesis.

Dynamic landscape of the local translation at activated synapses

The mammalian target of rapamycin (mTOR) signaling pathway is the central regulator of cap-dependent translation at the synapse. Disturbances in mTOR pathway have been associated with several neurological diseases, such as autism and epilepsy. RNA-binding protein FMRP, a negative regulator of translation initiation, is one of the key components of the local translation system. Activation and inactivation of FMRP occurs via phosphorylation by S6 kinase and dephosphorylation by PP2A phosphatase, respectively. S6 kinase and PP2A phosphatase are activated in response to mGluR receptor stimulation through different signaling pathways and at different rates. The dynamic aspects of this system are poorly understood. We developed a mathematical model of FMRP-dependent regulation of postsynaptic density (PSD) protein synthesis in response to mGluR receptor stimulation and conducted in silico experiments to study the regulatory circuit functioning. The modeling results revealed the possibility of generating oscillatory (cyclic and quasi-cyclic), chaotic and even hyperchaotic dynamics of postsynaptic protein synthesis as well as the presence of multiple attractors in a wide range of parameters of the local translation system. The results suggest that autistic disorders associated with mTOR pathway hyperactivation may be due to impaired proteome stability associated with the formation of complex dynamic regimes of PSD protein synthesis in response to stimulation of mGluR receptors on the postsynaptic membrane of excitatory synapses on pyramidal hippocampal cells.

The Role of Cytoplasmic mRNA Cap-Binding Protein Complexes in Trypanosoma brucei and Other Trypanosomatids.

Trypanosomatid protozoa are unusual eukaryotes that are well known for having unusual ways of controlling their gene expression. The lack of a refined mode of transcriptional control in these organisms is compensated by several post-transcriptional control mechanisms, such as control of mRNA turnover and selection of mRNA for translation, that may modulate protein synthesis in response to several environmental conditions found in different hosts. In other eukaryotes, selection of mRNA for translation is mediated by the complex eIF4F, a heterotrimeric protein complex composed by the subunits eIF4E, eIF4G, and eIF4A, where the eIF4E binds to the 5′-cap structure of mature mRNAs. In this review, we present and discuss the characteristics of six trypanosomatid eIF4E homologs and their associated proteins that form multiple eIF4F complexes. The existence of multiple eIF4F complexes in trypanosomatids evokes exquisite mechanisms for differential mRNA recognition for translation.

Effects of leucine-enriched essential amino acid and whey protein bolus dosing upon skeletal muscle protein synthesis at rest and after exercise in older women

SummaryBackground & aimsImpaired anabolic responses to nutrition and exercise contribute to loss of skeletal muscle mass with ageing (sarcopenia). Here, we tested responses of muscle protein synthesis (MPS), in the under represented group of older women, to leucine-enriched essential amino acids (EAA) in comparison to a large bolus of whey protein (WP).MethodsTwenty-four older women (65 ± 1 y) received (N = 8/group) 1.5 g leucine-enriched EAA supplements (LEAA_1.5), 6 g LEAA (LEAA_6) in comparison to 40 g WP. A primed constant I.V infusion of 13C6-phenylalanine was used to determine MPS at baseline and in response to feeding (FED) and feeding-plus-exercise (FED-EX; 6 × 8 unilateral leg extensions; 75%1-RM). We quantified plasma insulin/AA concentrations, leg femoral blood flow (LBF)/muscle microvascular blood flow (MBF), and anabolic signalling via immunoblotting.ResultsPlasma insulineamia and EAAemia were greater and more prolonged with WP than LEAA, although LEAA_6 peaked at similar levels to WP. Neither LEAA or WP modified LBF or MBF. FED increased MPS similarly in the LEAA_1.5, LEAA_6 and WP (P < 0.05) groups over 0–2 h, with MPS significantly higher than basal in the LEAA_6 and WP groups only over 0–4 h. However, FED-EX increased MPS similarly across all the groups from 0 to 4 h (P < 0.05). Only p-p70S6K1 increased with WP at 2 h in FED (P < 0.05), and at 2/4 h in FED-EX (P < 0.05).ConclusionsIn conclusion, LEAA_1.5, despite only providing 0.6 g of leucine, robustly (perhaps maximally) stimulated MPS, with negligible trophic advantage of greater doses of LEAA or even to 40 g WP. Highlighting that composition of EAA, in particular the presence of leucine rather than amount is most crucial for anabolism.

Influence of past injurious exercise on fiber type-specific acute anabolic response to resistance exercise in skeletal muscle

We investigated the influence of past injurious exercise on anabolic response of skeletal muscle fibers to resistance exercise (RE). Wistar rats were divided into exercise (E) and exercise-after-injury (I-E) groups. At age 10 wk, the right gastrocnemius muscle in each rat in the I-E group was subjected to strenuous eccentric contractions. Subsequently, RE was imposed on the same muscle of each rat at 14 wk of age in both groups. Peak joint torque and total force generation per body mass during RE were similar between the groups. Muscle protein synthesis (MPS) in the I-E group was higher than that in the E group 6 h after RE. Furthermore, levels of phospho-p70S6 kinase (Thr389) and phospho-ribosomal protein S6 (phospho-rpS6) (Ser240/244), a downstream target of p70S6 kinase, were higher in the I-E group than in the E group. For the anabolic response in each fiber type, the I-E group showed a higher MPS response in type IIb, IIa, and I fibers and a higher phospho-rpS6 response in type IIx, IIa, and I fibers than the E group. In the I-E group, the relative content of myosin heavy chain (MHC) IIa was higher and that of MHC IIb was lower than those in the E group. In addition, type IIa fibers showed a lower MPS response to RE than type IIb fibers in the I-E group. In conclusion, the past injurious exercise enhanced the MPS and phospho-rpS6 responses in type IIb, IIa, and I fibers and type IIx, IIa, and I fibers, respectively. NEW & NOTEWORTHY Past injurious exercise increased the muscle protein synthesis (MPS) response and mammalian target of rapamycin complex 1 (mTORC1) signaling activation to resistance exercise. In the responses of each fiber type, the past injurious exercise increased the MPS and phosphorylation ribosomal protein (Ser240/244) responses in type IIb, IIa, and I fibers and type IIx, IIa, and I fibers, respectively.

Short-term changes in diet composition do not affect in vivo hepatic protein synthesis in rats.

Protein synthesis is critical to protein homeostasis (proteostasis), and modifications in protein synthesis influence lifespan and the development of comorbidities associated with obesity. In the present study, we examined the acute response of liver protein synthesis to either high-fat or high-sucrose diets in order to elucidate nutrient-mediated regulation of hepatic protein synthesis in the absence of body fat accumulation. Total and endoplasmic reticulum-associated protein syntheses were assessed by use of the stable isotope, deuterium oxide (2H2O), in rats provided a control diet or diets enriched in polyunsaturated fat, saturated fat, or sucrose for 2, 4, or 7 days. The three experimental diets increased hepatic triglycerides 46-91% on day 7 and fasting insulin levels 83-117% on day 7, but did not result in differences in body weight when compared with control ( n = 6/diet/time). The fraction of newly synthesized proteins in total liver lysates and microsomes was not significantly different among dietary groups ( n = 3/diet/time). To determine whether the experimental diets provoked a transcriptional response to enhance the capacity for protein synthesis, we also measured a panel of genes linked to amino acid transport, synthesis, and processing. There were no significant differences in any of the genes measured among groups. Therefore, dietary treatments that have been linked to impaired proteostasis and that promote hepatic steatosis and insulin resistance, did not result in significant changes in total or ER-associated protein synthesis in the liver over a 7-day period.

Skeletal Muscle Remodeling in Response to Eccentric vs. Concentric Loading: Morphological, Molecular, and Metabolic Adaptations.

Skeletal muscle contracts either by shortening or lengthening (concentrically or eccentrically, respectively); however, the two contractions substantially differ from one another in terms of mechanisms of force generation, maximum force production and energy cost. It is generally known that eccentric actions generate greater force than isometric and concentric contractions and at a lower metabolic cost. Hence, by virtue of the greater mechanical loading involved in active lengthening, eccentric resistance training (ECC RT) is assumed to produce greater hypertrophy than concentric resistance training (CON RT). Nonetheless, prevalence of either ECC RT or CON RT in inducing gains in muscle mass is still an open issue, with some studies reporting greater hypertrophy with eccentric, some with concentric and some with similar hypertrophy within both training modes. Recent observations suggest that such hypertrophic responses to lengthening vs. shortening contractions are achieved by different adaptations in muscle architecture. Whilst the changes in muscle protein synthesis in response to acute and chronic concentric and eccentric exercise bouts seem very similar, the molecular mechanisms regulating the myogenic adaptations to the two distinct loading stimuli are still incompletely understood.Thus, the present review aims to, (a) critically discuss the literature on the contribution of eccentric vs. concentric loading to muscular hypertrophy and structural remodeling, and, (b) clarify the molecular mechanisms that may regulate such adaptations.We conclude that, when matched for either maximum load or work, similar increase in muscle size is found between ECC and CON RT. However, such hypertrophic changes appear to be achieved through distinct structural adaptations, which may be regulated by different myogenic and molecular responses observed between lengthening and shortening contractions.

Effects of graded removal of lysine from an intravenously infused amino acid mixture on lactation performance and mammary amino acid metabolism in lactating goats

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies.

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.

α7β1 Integrin regulation of gene transcription in skeletal muscle following an acute bout of eccentric exercise.

The α7β1 integrin is concentrated at the costameres of skeletal muscle and provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We previously demonstrated that expression of the α7BX2 integrin subunit (MCK:α7BX2) preserves muscle integrity and enhances myofiber cross-sectional area following eccentric exercise. The purpose of this study was to utilize gene expression profiling to reveal potential mechanisms by which the α7BX2-integrin contributes to improvements in muscle growth after exercise. A microarray analysis was performed using RNA extracted from skeletal muscle of wild-type or transgenic mice under sedentary conditions and 3 h following an acute bout of downhill running. Genes with false discovery rate probability values below the cutoff of P < 0.05 (n = 73) were found to be regulated by either exercise or transgene expression. KEGG pathway analysis detected upregulation of genes involved in endoplasmic reticulum protein processing with integrin overexpression. Targeted analyses verified increased transcription of Rpl13a, Nosip, Ang, Scl7a5, Gys1, Ndrg2, Hspa5, and Hsp40 as a result of integrin overexpression alone or in combination with exercise (P < 0.05). A significant increase in HSPA5 protein and a decrease in CAAT-enhancer-binding protein homologous protein (CHOP) were detected in transgenic muscle (P < 0.05). In vitro knockdown experiments verified integrin-mediated regulation of Scl7a5 The results from this study suggest that the α7β1 integrin initiates transcription of genes that allow for protection from stress, including activation of a beneficial unfolded protein response and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle as a result of engagement in eccentric exercise.

Investigation of protein synthesis in Drosophila larvae using puromycin labelling.

ABSTRACTTranslational control of gene expression is an important regulator of growth, homeostasis and aging in Drosophila. The ability to measure changes in protein synthesis in response to genetic and environmental cues is therefore important in studying these processes. Here we describe a simple and cost-effective approach to assay protein synthesis in Drosophila larval cells and tissues. The method is based on the incorporation of puromycin into nascent peptide chains. Using an ex vivo approach, we label newly synthesized peptides in larvae with puromycin and then measure levels of new protein synthesis using an anti-puromycin antibody. We show that this method can detect changes in protein synthesis in specific cells and tissues in the larvae, either by immunostaining or western blotting. We find that the assay reliably detects changes in protein synthesis induced by two known stimulators of mRNA translation – the nutrient/TORC1 kinase pathway and the transcription factor dMyc. We also use the assay to describe how protein synthesis changes through larval development and in response to two environmental stressors – hypoxia and heat shock. We propose that this puromycin-labelling assay is a simple but robust method to detect protein synthesis changes at the levels of cells, tissues or whole body in Drosophila.