Translation Initiation Factor Activation Research Articles

Antibacterial mechanism of inosine against Alicyclobacillus acidoterrestris.

Inosine could potentially become a novel antibacterial agent against Alicyclobacillus acidoterrestris as low doses of inosine can prevent its contamination. However, until now the antibacterial mechanism of inosine targeting A. acidoterrestris is still unknown. In this study, to unravel the mechanism of inosine against A. acidoterrestris puzzle, the effects of inosine on bacterial surface hydrophobicity, intracellular protein content, cell membrane damage extent, and permeability of the A. acidoterrestris were investigated. The results showed that inosine can effectively inhibit the growth and reproduction of A. acidoterrestris by destroying the integrity of cell membrane and increasing its permeability, causing the leakage of intracellular nutrients. Furthermore, the interaction networks of inosine target proteins were analyzed. The interaction networks further revealed that damage to bacterial cell membranes might be relevant to inosine's effect on bacterial DNA replication and cell energy metabolism through regulating nucleotide synthesis and metabolism and the activity of translation initiation factors. Finally, the antibacterial mechanism of inosine against A. acidoterrestris was proposed.

The Integrated Stress Response Is Tumorigenic and Constitutes a Therapeutic Liability in Somatotroph Adenomas.

Somatotroph adenomas are the leading cause of acromegaly, with the nearly sparsely granulated somatotroph subtype belonging to high-risk adenomas, and they are less responsive to medical treatment. The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. In this study, we identified the characteristic profiling of the integrated stress response in translocation and translation initiation factor activity in somatotroph adenomas, normal pituitary, or other adenoma subtypes through proteomics. Immunohistochemistry exhibited the differential significance and the priority of eukaryotic translation initiation factor 2β (EIF2β) in somatotroph adenomas compared with gonadotroph and corticotroph adenomas. Differentially expressed genes based on the level of EIF2β in somatotroph adenomas were revealed. MetaSape pathways showed that EIF2β was involved in regulating growth and cell activation, immune system, and extracellular matrix organization processes. The correlation analysis showed Spearman correlation coefficients of r = 0.611 (p < 0.001) for EIF2β and eukaryotic translation initiation factor 2 alpha kinase 1 (HRI), r = 0.765 (p < 0.001) for eukaryotic translation initiation factor 2 alpha kinase 2 (PKR), r = 0.813 (p < 0.001) for eukaryotic translation initiation factor 2 alpha kinase 3 (PERK), r = 0.728 (p < 0.001) for GCN2, and r = 0.732 (p < 0.001) for signal transducer and activator of transcription 3 (STAT3). Furthermore, the invasive potential in patients with a high EIF2β was greater than that in patients with a low EIF2β (7/10 vs. 4/18, p = 0.038), with a lower immune-cell infiltration probability (p < 0.05). The ESTIMATE algorithm showed that the levels of activation of the EIF2 pathway were negatively correlated with the immune score in somatotroph adenomas (p < 0.001). In in vitro experiments, the knockdown of EIF2β changed the phenotype of somatotroph adenomas, including cell proliferation, migration, and the secretion ability of growth hormone/insulin-like growth factor-1. In this study, we demonstrate that the ISR is pivotal in somatotroph adenomas and provide a rationale for implementing ISR-based regimens in future treatment strategies.

Global proteomic analysis of preimplantational ovine embryos produced in vitro.

The present study was conducted to characterize the major proteome of preimplantation (D6) ovine embryos produced in vitro. COCs were aspirated from antral follicles (2-6mm), matured and fertilized in vitro and cultured until day six. Proteins were extracted separately from three pools of 45 embryos and separately run in SDS-PAGE. Proteins from each pool were individually subjected to in-gel digestion followed by LC-MS/MS. Three 'raw files' and protein lists were produced by Pattern Lab software, but only proteins present in all three lists were used for the bioinformatics analyses. There were 2,262 proteins identified in the 6-day-old ovine embryos, including albumin, zona pellucida glycoprotein 2, 3 and 4, peptidyl arginine deiminase 6, actin cytoplasmic 1, gamma-actin 1, pyruvate kinase, heat shock protein 90 and protein disulfide isomerase, among others. Major biological processes linked to the sheep embryo proteome were translation, protein transport and protein stabilization, and molecular functions, defined as ATP binding, oxygen carrier activity and oxygen binding. There were 42 enriched functional clusters according to the 2,147 genes (UniProt database). Ten selected clusters with potential association with embryo development included translation, structural constituent of ribosomes, ribosomes, nucleosomes, structural constituent of the cytoskeleton, microtubule-based process, translation initiation factor activity, regulation of translational initiation, cell body and nucleotide biosynthetic process. The most representative KEEG pathways were ribosome, oxidative phosphorylation, glutathione metabolism, gap junction, mineral absorption, DNA replication and cGMP-PKG signalling pathway. Analyses of functional clusters clearly showed differences associated with the proteome of preimplantation (D6) sheep embryos generated after in vitro fertilization in comparison with in vivo counterparts (Sanchez etal.,2021; https://doi.org/10.1111/rda.13897), confirming that the quality of in vitro derived blastocysts are unlike those produced in vivo. The present study portrays the first comprehensive overview of the proteome of preimplantational ovine embryos grown in vitro.

Translational Regulation by hnRNP H/F Is Essential for the Proliferation and Survival of Glioblastoma.

Simple SummaryDeveloping effective treatments for glioblastoma (GBM), a highly aggressive brain tumor that is resistant to current therapies, is an urgent medical need that can be addressed by the in-depth study of basic biology for the identification of relevant targets. The aim of the present study was to investigate the molecular mechanisms underlying the deregulation of protein synthesis associated with GBM progression and resistance to treatments. Our present work demonstrates the role of the RNA-binding proteins hnRNP H/F as key players in the control of protein synthesis in GBM through different overlapping mechanisms. Furthermore, our results show that hnRNP H/F potentiate cellular processes underlying the aggressive and resistant phenotype of GBMs, thus indicating hnRNP H/F as a potential target for therapeutic intervention.Deregulation of mRNA translation is a widespread characteristic of glioblastoma (GBM), aggressive malignant brain tumors that are resistant to conventional therapies. RNA-binding proteins (RBPs) play a critical role in translational regulation, yet the mechanisms and impact of these regulations on cancer development, progression and response to therapy remain to be fully understood. Here, we showed that hnRNP H/F RBPs are potent regulators of translation through several mechanisms that converge to modulate the expression and/or the activity of translation initiation factors. Among these, hnRNP H/F regulate the phosphorylation of eIF4E and its translational targets by controlling RNA splicing of the A-Raf kinase mRNA, which in turn modulates the MEK-ERK/MAPK signaling pathway. The underlying mechanism involves RNA G-quadruplex (RG4s), RNA structures whose modulation phenocopies hnRNP H/F translation regulation in GBM cells. Our results highlighted that hnRNP H/F are essential for key functional pathways regulating proliferation and survival of GBM, highlighting its targeting as a promising strategy for improving therapeutic outcomes.

The E3 ubiquitin ligase Itch deficiency promotes antigen-driven B-cell responses in mice.

Deficiency of Itch, an E3 ubiquitin ligase, usually induced severe systemic and progressive autoimmune disease. The Itch function is well studied in T cells but not in B cells. We hypothesize that B-cell-specific Itch deficiency promoted antigen-induced B-cell activation and antibody-expressing plasma cell (PC) production. We found that unlike Itch KO, Itch cKO (CD19cre Itchf/f ) mice did not demonstrated a significant increase in the sizes of spleens and LNs, antibody level, and base mutation of antibody gene. However, in line with the fact that Itch expression decreased in GC B cells, PCs, and plasmablast (PB)-like SP 2/0 cells, Itch deficiency promoted B-cell activation and antibody production induced by antigens including lipopolysaccharide (LPS) and sheep red blood cells (SRBCs). Mechanistically, we found that Itch deficiency promotes antigen-induced cytokine production because Itch controls the proteins (e.g., eIF3a, eIF3c, eIF3h) with translation initiation factor activity. Altogether, our data suggest that Itch deficiency promotes antigen-driven B-cell response. This may provide hints for Itch-targeted treatment of patients with autoimmune disease.

12 Effect of long-term leucine supplementation on muscle protein synthesis in a pig model of neonatal growth.

Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. Both nutrient intolerance and the risk of developing feeding-related pathologies have led to the implementation of feeding protocols below nutrient requirements, that often result in periods of malnourishment with potential long-term adverse consequences for the neonate. A parallel situation when early growth is compromised is encountered during early weaning of pigs, caused by a reduction in food intake due to stress, environmental changes, and a shift from liquid to solid diet, in conjunction with an increase in nutrient requirements. The resulting growth retardation and mortality represents a major production loss for the swine industry. In the neonate, the primary driving force for the elevated rate of protein accretion is the capacity to attain high rates of protein synthesis after feeding. Studies in neonatal pigs have shown that the sharp increase in muscle protein synthesis after eating is mediated by the rise in amino acids and insulin, which activate independent signaling pathways that converge at mammalian target of rapamycin complex 1 (mTORC1), leading to the activation of key regulators of translation. Leucine is the most effective single amino acid in triggering translation initiation factor activation. Pulsatile administration of leucine during continuous orogastric feeding of a milk replacer during 21 days enhances muscle protein synthesis by stimulating phosphorylation of mTORC1 and its downstream effectors, including ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4 binding protein 1 (4EBP1), while downregulates both cellular autophagy systems and insulin-independent pathways involved in protein catabolism. Prolonged leucine supplementation under protein and energy restriction increases the activation of S6K1 and 4EBP1 but does not improved protein synthesis and body growth in neonates. Whether the anabolic effects of leucine can be sustained chronically to promote lean growth requires further study.

EIF4E-Dependent Translational Control: A Central Mechanism for Regulation of Pain Plasticity.

Translational control of gene expression has emerged as a key mechanism in regulating different forms of long-lasting neuronal plasticity. Maladaptive plastic reorganization of peripheral and spinal nociceptive circuits underlies many chronic pain states and relies on new gene expression. Accordingly, downregulation of mRNA translation in primary afferents and spinal dorsal horn neurons inhibits tissue injury-induced sensitization of nociceptive pathways, supporting a central role for translation dysregulation in the development of persistent pain. Translation is primarily regulated at the initiation stage via the coordinated activity of translation initiation factors. The mRNA cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E), is involved in the recruitment of the ribosome to the mRNA cap structure, playing a central role in the regulation of translation initiation. eIF4E integrates inputs from the mTOR and ERK signaling pathways, both of which are activated in numerous painful conditions to regulate the translation of a subset of mRNAs. Many of these mRNAs are involved in the control of cell growth, proliferation, and neuroplasticity. However, the full repertoire of eIF4E-dependent mRNAs in the nervous system and their translation regulatory mechanisms remain largely unknown. In this review, we summarize the current evidence for the role of eIF4E-dependent translational control in the sensitization of pain circuits and present pharmacological approaches to target these mechanisms. Understanding eIF4E-dependent translational control mechanisms and their roles in aberrant plasticity of nociceptive circuits might reveal novel therapeutic targets to treat persistent pain states.

Abstract B13: EGF/Ras/Erk signaling controls growth and proliferation through regulation of tRNA synthesis

Abstract Ras signaling promotes growth and proliferation in many tissues throughout animal development. An important challenge is to identify how the Ras-Erk pathway alters cellular metabolism to drive growth. Here we report on the control of tRNA synthesis as growth effector of EGF/Ras/Erk signaling in Drosophila. I find that overexpression of oncogenic Ras (RasV12) leads to increased mRNA translation and protein content in Drosophila S2 cells, suggesting that Ras may promote growth through enhanced protein synthesis. The conventional view is that the Ras pathway functions by controlling translation initiation factor activity. However I have identified an alternate mechanism involving control of tRNA synthesis. My data suggest that overexpression of RasV12 or the activated versions of EGFR and the Raf1 in wing imaginal discs increases tRNA synthesis. Similarly, expression of RasV12 in S2 cells increases tRNA levels, while blocking Ras/Erk signaling using the MEK inhibitor, U0126 or RNAi reduces tRNA synthesis. We previously identified the RNA polymerase III (Pol III) factor, Brf, as regulator of cell and tissue growth in Drosophila. Here we show that knockdown of either Brf blocks the effects of Ras signaling on growth and proliferation in larval wing imaginal discs, adult midgut progenitor cells and adult intestinal stem cells. Several transcription factors have been shown to link Ras signaling to changes in mRNA expression and growth. I have identified Myc is required but not sufficient for Ras-induced cell proliferation and growth through tRNA synthesis. Previously we have shown that TOR signaling regulates protein synthesis through RNA Pol III repressor, Maf1. I found that Maf1 RNAi increases tRNA synthesis in S2 cells. In addition, the decrease in tRNA synthesis induced by the MEK inhibitor is blocked in the presence of Maf1 RNAi suggesting Maf1 is downstream of Ras signaling pathway. My data point to control of tRNA synthesis possibly through Myc and/or Maf1 as a new mechanisms by which Ras signaling enhances protein synthesis to promote cell and tissue growth. Citation Format: Shrivani Sriskanthadevan-Pirahas, Savraj S. Grewal. EGF/Ras/Erk signaling controls growth and proliferation through regulation of tRNA synthesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B13.

Comparative Proteomic Profiling of Refractory/Relapsed Multiple Myeloma Patient Plasma Cells Reveals Biomarkers and Pathways Involved in Bortezomib-Based-Therapy Resistance

Introduction: Despite the new drugs introduced to the treatment of multiple myeloma (MM) the disease remains incurable due to increasing resistance to therapy. Understanding biology of disease and identification of biomarkers of resistance is a key research challenge and may enable the individualization of the treatment.Objective: The goal of this study was comparative proteomic profiling of pre-treatment plasma cells (PCs) from patients with refractory/relapsed MM (RRMM) subsequently treated with PAD (Bortezomib, Adriamycin, Dexamethasone) to identify biomarkers and signaling pathways differing PCs from patients who achieved CR and those with lower response. We wanted to expand recently published (Dytfeld et al, 2015) catalog of proteins and pathways involved in resistance to bortezomib-based therapy in newly-diagnosed multiple myeloma (NDMM).Material and Methods: Comparative proteome analysis was performed on purified by negative selection (EasySep) pre-treatment PCs from 24 patients with RRMM qualified for PAD. PCs acquired from bone marrow specimens afterobtaining informed consent from patients were lysed, and protein was isolated. Trypsin digested peptides were labeled with iTRAQ (4-plex) reagents and subsequently analyzed using Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to the chromatograph Dionex 3000 Ultimate nanoLC (Thermo Scientific). Proteins which accumulation in the analyzed subgroups differed by at least 50% were considered to differentiate. Potential biomarkers were validated using ELISA in the patients' sera. The dysregulated proteins were subjected to two bioinformatic tools: PANTHER and DAVID, for identifying enriched signaling pathways or networks. Only these suggested by both tools were considered significant.Results and discussion: Out of 3821 proteins identified (FDR <1%), 325 showed significant differences in accumulation between patients who after treatment with PAD achieved CR and patients with worse response. TXNDC5 (peroxiredoxin activity) and PSME1 (proteasome component) found elevated in PCs from refractory patients were confirmed by ELISA in patients' sera (TXNDC5: 110.1pg/mL vs. 40.47, p =0.03; PSME1: 222.02 pg/ml vs. 141.99 pg/mL, p =0.03). Based on the log of p-value from Fisher's test and number of identified differential proteins, the most affected pathways constitute: proteasome pathway (55 proteins; p =3.5x10-19), oxidative phosphorylation (43 proteins; p =4.5x10-11), ribosome proteins (68 proteins; p =1.1x10-47), spliceosome proteins (46 proteins; p =1,5x10-13), translation initiation factor activity (24 proteins; p =1.16x10-11) and peroxiredoxin activity (7 proteins; p =3.7x10-6). All of them were up-regulated in resistant patients. The most affected pathways were previously identified in NDMM treated subsequently with VDD and RVD. Elevated proteasome activity in resistant patients may be a result of affected expression of proteins related to oxidative phosphorylation and enhanced production of reactive oxygen species connected with up-regulation of peroxisome activity proteins. As a consequence bortezomib may not inhibit proteasome sufficiently, thus prevents apoptosis. An alteration in the stoichiometry of components of the proteasome, ribosome, and spliceosome is the effect of genomic instability and thereby most cancers exhibit loss of genes responsible for tumor suppressor (Nijhawan et al, 2012).Conclusion: We indicated biomarkers of resistance which may be a potential therapeutic targets and basis for individualization of MM therapy. We also showed pathways involved in resistance to bortezomib-based therapy in RRMM that confirms our previous finding in NDMM. Further studies are necessary to explore these pathways and define their role in multiple myeloma treatment.Study was supported by research grant funded by Janssen Cilag. DisclosuresDytfeld:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowiak:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: institutional funding for support of clinical trial conduct, Speakers Bureau; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Leucine Attenuates the Endotoxin‐induced Reduction in Skeletal Muscle Protein Synthesis in Neonatal Pigs

Sepsis decreases protein synthesis (PS) in skeletal muscle and mitigates the anabolic response to Leucine (Leu) in mature animals. To determine if supplemental Leu can stimulate muscle PS in the neonate during endotoxemia, overnight fasted neonatal pigs were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic substrate clamps. Leu or saline was infused during the last h. PS and translation initiation factor activation were determined in skeletal muscle. LPS decreased protein synthesis by 36% in longissimus dorsi (LD), 28% in gastrocnemius (Gast) and 38% in soleus muscles compared to controls, but Leu infusion in the LPS group increased protein synthesis by 84% in LD, 81% in Gast and 83% in soleus compared to LPS infusion alone. Leu infusion alone compared to controls also increased protein synthesis in the LD, Gast and Soleus muscles. Leu infusion in both healthy and endotoxemic piglets compared to non-Leu infused piglets increased 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles. These findings suggest that Leu supplementation can attenuate the reduction in muscle PS induced by endotoxemia by stimulating mTORC1-dependent translation in the neonate. (Supported by NIH AR444474, USDA/ARS 6250-51000-055, NIH HD072891, and USDA NIFA 2013-67015-20438)

Structure–activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein–protein interaction inhibitors

Protein–protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein–protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities.

Changes in Translational Control after Pro-Apoptotic Stress

In stressed cells, a general decrease in the rate of protein synthesis occurs due to modifications in the activity of translation initiation factors. Compelling data now indicate that these changes also permit a selective post-transcriptional expression of proteins necessary for either cell survival or completion of apoptosis when cells are exposed to severe or prolonged stress. In this review, we summarize the modifications that inhibit the activity of the main canonical translation initiation factors, and the data explaining how certain mRNAs encoding proteins involved in either cell survival or apoptosis can be selectively translated.

Novel events in the molecular regulation of muscle mass in critically ill patients

Critically ill patients experience marked skeletal muscle atrophy, but the molecular mechanisms responsible for this are largely unresolved. Therefore, we investigated key genes and proteins, identified from cell and animal studies to control protein synthesis and breakdown, in vastus lateralis biopsy samples obtained from 10 patients and 10 age- and sex-matched healthy controls. Muscle cytokines IL-6 and TNF-α mRNA were higher in patients than in controls(6.5-fold; P < 0.001 and 2-fold; P < 0.01). From the perspective of muscle protein breakdown, muscle-specific E3-ligases (MAFbx and MuRF1) were higher in patients at mRNA (4.5-fold; P < 0.05 and 2.5-fold; P < 0.05) and protein (5-fold; P < 0.001 and 4.5-fold; P < 0.001) level. Furthermore, 20S proteasome mRNA and protein were higher in patients (5-fold; P < 0.001 and 2.5-fold; P < 0.01). Cathepsin-L mRNA was 2-fold higher (P < 0.01), whilst calpain-3 mRNA(2-fold; P < 0.01) and protein (4-fold; P < 0.01)were lower inpatients. Another novel observation was the 3-fold (P < 0.05) and 8.5-fold (P < 0.001) higher expression of myostatin mRNA and protein in patients. Widespread dephosphorylation (inactivation) of proteins regulating translation initiation factor activation and protein synthesis (Akt1, GSK3α,β, mTOR, p70S6K and 4E-BP1) was observed in patients, which was paralleled by increases in their mRNAs. Finally, PDK4 mRNA and protein was 2-fold (P < 0.05) and 2.6-fold (P < 0.01), respectively, higher inpatients. In conclusion, we showed comprehensive alterations in molecular events thought to reduce muscle mass and carbohydrate (CHO) oxidation in critically ill patients. Nevertheless,these catabolic events were matched by a cellular programme of anabolic restoration at the transcriptional level. This shows a high molecular plasticity in the muscle of patients, and strategies to preserve muscle mass and metabolic function should focus on maintaining Akt phosphorylation and inhibiting myostatin expression.C

Transcriptomic Analysis of the Osmotic and Reproductive Remodeling of the Female Rat Supraoptic Nucleus

The supraoptic nucleus (SON) of the hypothalamus is an important integrative brain structure that coordinates responses to perturbations in water balance and regulates maternal physiology through the release of the neuropeptide hormones vasopressin and oxytocin into the circulation. Both dehydration and lactation evoke a dramatic morphological remodeling of the SON, a process known as function-related plasticity. We hypothesize that some of the changes seen in SON remodeling are mediated by differential gene expression, and have thus used microarrays to document global changes in transcript abundance that accompany chronic dehydration in female rats, and in lactation. In situ hybridization analysis has confirmed the differential expression of three of these genes, namely TNF-induced protein 6, gonadotropin-inducible transcription factor 1, and ornithine decarboxylase antizyme inhibitor 1. Comparison of differential gene expression patterns in male and female rats subjected to dehydration and in lactating rats has enabled the identification of common elements that are significantly enriched in gene classes with particular functions. Two of these are related to the requirement for increased protein synthesis and hormone delivery in the physiologically stimulated SON (translation initiation factor activity and endoplasmic reticulum-Golgi intermediate compartment, respectively), whereas others are consistent with the concept of SON morphological plasticity (collagen fibril organization, extracellular matrix organization and biogenesis, extracellular structure organization and biogenesis, and homophilic cell adhesion). We suggest that the genes coordinately regulated in the SON as a consequence of dehydration and lactation form a network that mediates the plastic processes operational in the physiologically activated SON.

Leucine and α-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs , ,

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, α-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 μmol·kg−1·h−1 for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1·eIF4E complex, and greater (P < 0.05) active eIF4G·eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

Differential Regulation of Protein Synthesis and mTOR Signaling in Skeletal Muscle and Visceral Tissues of Neonatal Pigs after a Meal

Neonates have a high rate of growth driven by elevated rates of skeletal muscle protein synthesis. Feeding stimulates protein synthesis but the time course of the changes in different tissues has not been determined. The aim of this study was to evaluate the time course of the changes in protein synthesis rates and translation initiation factor activation in skeletal muscle and visceral tissues after a meal. Neonatal pigs (n=36; 5–7 day old) received a meal providing one‐sixth of daily requirements and were sacrificed at 0, 30, 60, 90, 120, or 240 min after feeding. Protein synthesis increased 30 to 120 min in glycolytic muscles, 30 to 90 min in mixed fiber type muscle and did not increase in oxidative muscle post‐feeding. Protein synthesis increased in liver 30 to 120 min, jejunum 30 to 60 min and pancreas 30 min post‐feeding and did not increase in kidney and heart. Eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) and rp S6 kinase 1 (S6K1) phosphorylation increased 30 to 90 min in all tissues. These results suggest that feeding stimulates mTOR signaling in skeletal muscle and visceral tissues. However, mTOR activation alone was not sufficient to stimulate protein synthesis in all tissues. (Supported by NIH grants AR‐44474 and AR‐51563 and USDA/ARS 6250510000‐33).Grant Funding Source: NIH grants AR‐44474 and AR‐51563 and USDA/ARS 6250510000‐33

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

Effect of different leucine supplementation in low protein diet on Protein Synthesis and Activation of Translation Initiation Factors of Weaned Piglets

Acute administration of L‐leucine stimulates protein synthesis in skeletal muscle of young pigs. However, little is known about a role for the chronic enteral feeding of leucine in regulating the activity of translation initiation factors, nutrient utilization, or growth of the neonate. Eighteen 21‐day crossbred pigs with an average live body weight 8 kg were assigned randomly to one of three groups and feed with low protein dietaries (16% crude protein) containing 1.3%, 1.6%, or 1.9% leucine which meets the marginal, adequate and excess leucine requirement of piglets respectively. At 35 d old, protein synthesis was measured and blood samples were obtained at 15, 30, 45min after infusion for metabolite analysis and the abundance and phosphorylation state of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E‐binding protein‐1 (4E‐BP1) were measured in longissimus muscle and liver. The results shows that excess leucine could increase (P &lt; 0.05) the fractional synthesis rate (FSR) of heart, posterior small intestine, kidney, liver, spleen, stomach and pancreas compared with pigs adequate and marginal group. Data in the present study also show that excess leucine could increase positive translation initiation factors activity in liver and muscle compared with marginal leucine group. These findings suggest that, despite the activation of positive translation initiation factors, adequate availability of essential amino acids may be necessary for leucine to stimulate muscle protein synthesis in young pigs.

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.