Activation Of Eukaryotic Initiation Factor Research Articles

Proteomic Analysis of Glucose-Induced Cardiac Myoblasts and the Potential Role of mir-92b-5p in Regulating Sarcomere Proteins Under a Hyperglycemic Environment.

Diabetes mellitus, a common metabolic disorder that causes high blood glucose, is due to impaired insulin secretion. Prolonged high blood sugar is associated with heart disease. Many proteins are involved in metabolic pathways and contractility of cardiac cells regulate cardiac hypertrophy, altering normal cardiac physiology and function. Moreover, microRNAs are essential regulators of these proteins. Thus, there is a need to study the protein and microRNA alterations in cardiomyocytes to better understand the mechanisms activated during cardiac stress. The study aims to profile differentially expressed sarcomere proteins in H9C2 cell lines under high glucose conditions compared with normal conditions, along with the identification of miRNAs regulating these proteins. Cardiac myoblast cell lines were treated with D-Glucose at three concentrations (10 mM, 25 mM, and 50 mM). Total cell protein was analyzed by Tandem Mass spectrometry Nano LCMS/ MS. Furthermore, next-generation sequencing data were analyzed for detecting microRNAs regulating cardiac cell protein expression. Bioinformatics databases such as Uniprot, Ingenuity Pathway Analysis (IPA), PANTHER, and Target scan were used. The Nano LC-MS/MS analysis showed 2891 protein, 1351 protein groups, and 4381 peptide groups in both glucose-treated and control samples. Most proteins were metabolite interconversion enzymes, translation proteins, and proteins regulating the cytoskeleton. IPA analysis revealed differentially expressed proteins involved in EIF2 signaling, actin cytoskeleton signaling, cardiac fibrosis, and cell death. Moreover, the proteins troponin, tropomyosin, myosin, alpha-actin, and ATP synthase were found to be downregulated, thus responsible for altering sarcomere protein expression. Rno-mir-92b-5p was observed to be highly upregulated at 50 mM. Its target genes namely TPM2, ATP1A2, and CORO1C were mostly components of the sarcomere complex and its regulators. A combination of proteomic profile and microRNA profile of hyperglycemic cells provides an insight into advanced therapeutics. Our study has highlighted the role of sarcomere proteins, activation of Eukaryotic Initiation Factor 2 (EIF2) signaling, and suppression of actin cytoskeleton signaling in the pathophysiology of cardiomyopathy. MiR-92b-5p has an important role in regulating sarcomere protein complex activated.

Melatonin Inhibits Glucose-Induced Apoptosis in Osteoblastic Cell Line Through PERK-eIF2α-ATF4 Pathway.

Osteoporosis is a common disease resulting in deteriorated microarchitecture and decreased bone mass. In type 2 diabetes patients, the incidence of osteoporosis is significantly higher accompanied by increased apoptosis of osteoblasts. In this study, using the osteoblastic cell line MC3T3-E1, we show that high glucose reduces cell viability and induces apoptosis. Also, high glucose leads to endoplasmic reticulum (ER) stress (ERS) via an increase in calcium flux and upregulation of the ER chaperone binding immunoglobulin protein (BiP). Moreover, it induces post-translational activation of eukaryotic initiation factor 2 alpha (eIF2α) which functions downstream of PKR-like ER kinase (PERK). This subsequently leads to post-translational activation of the transcription factor 4 (ATF4) and upregulation of C/EBP-homologous protein (CHOP) which is an ER stress-induced regulator of apoptosis, as well as downstream effectors DNAJC3, HYOU1, and CALR. Interestingly, melatonin treatment significantly alleviates the high-glucose induced changes in cell growth, apoptosis, and calcium influx by inhibiting the PERK-eIF2α-ATF4-CHOP signaling pathway. Additionally, the MC3T3-E1 cells engineered to express a phosphodead eIF2α mutant did not show high glucose induced ER stress, confirming that melatonin protects osteoblasts against high-glucose induced changes by decreasing ER-stress induced apoptosis by impacting the PERK-eIF2α-ATF4-CHOP signaling pathway. The protective of melatonin against high glucose-induced ER stress and apoptosis was attenuated when the cells were pre-treated with a melatonin receptor antagonist, indicating that the effect of melatonin was mediated via the melatonin receptors in this context. These findings lay the provide mechanistic insights of melatonin’s protective action on osteoblasts and will be potentially be useful in ongoing pre-clinical and clinical studies to evaluate melatonin as a therapeutic option for diabetic osteoporosis.

BCR-ABL tyrosine kinase inhibition induces metabolic vulnerability by preventing the integrated stress response in K562 cells

The integrated stress response (ISR) is a cellular process that is characterized by activation of eukaryotic initiation factor (eIF)2α kinases and subsequent induction of activating transcription factor (ATF)4. The ISR plays an important role in protecting cells from tumor-related metabolic stresses, such as nutrient deprivation and perturbed proteostasis. Here, we showed that disruption of the ISR, together with increased cellular stress vulnerability, was produced by pharmacological inhibition of BCR-ABL, the oncogenic driver in chronic myeloid leukemia (CML). Treatment of CML-derived K562 cells with BCR-ABL tyrosine kinase inhibitors, including imatinib, dasatinib, nilotinib and ponatinib, prevented activation of eIF2α kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control nonderepressible 2, and downstream ATF4 induction during metabolic stress. Prevention of ATF4 induction likely occurred as a result of the combinatorial suppression of the eIF2α kinase and phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathways. In addition, we found that pharmacological inhibition of PERK mimicked BCR-ABL inhibition to enhance apoptosis induction under stress conditions. These findings indicate that the ISR is under the control of BCR-ABL and may foster adaptation to tumorigenic stresses in CML cells.

Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A.

Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation - more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target.

Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.

SUMO-2 promotes mRNA translation by enhancing interaction between eIF4E and eIF4G.

Small ubiquitin-like modifier (SUMO) proteins regulate many important eukaryotic cellular processes through reversible covalent conjugation to target proteins. In addition to its many well-known biological consequences, like subcellular translocation of protein, subnuclear structure formation, and modulation of transcriptional activity, we show here that SUMO-2 also plays a role in mRNA translation. SUMO-2 promoted formation of the active eukaryotic initiation factor 4F (eIF4F) complex by enhancing interaction between Eukaryotic Initiation Factor 4E (eIF4E) and Eukaryotic Initiation Factor 4G (eIF4G), and induced translation of a subset of proteins, such as cyclinD1 and c-myc, which essential for cell proliferation and apoptosis. As expected, overexpression of SUMO-2 can partially cancel out the disrupting effect of 4EGI-1, a small molecule inhibitor of eIF4E/eIF4G interaction, on formation of the eIF4F complex, translation of the cap-dependent protein, cell proliferation and apoptosis. On the other hand, SUMO-2 knockdown via shRNA partially impaired cap-dependent translation and cell proliferation and promoted apoptosis. These results collectively suggest that SUMO-2 conjugation plays a crucial regulatory role in protein synthesis. Thus, this report might contribute to the basic understanding of mammalian protein translation and sheds some new light on the role of SUMO in this process.

Activation of eIF2α signaling cascade is associated with testosterone-induced cell apoptosis in INS-1 cells.

Hyperandrogenemia is associated with insulin resistance and type 2 diabetes in women with polycystic ovary syndrome raising the possibility that androgen receptor signaling pathway plays an important role in the development and progression of β-cell dysfunction. Testosterone is the major circulating androgen in women. In this study, we investigated the effect of testosterone on INS-1 cells to find whether excess androgen could produce endoplasmic reticulum (ER) stress thereby contributing to β-cell dysfunction. The role of testosterone in INS-1 cell apoptosis was detected by flow cytometry and electron microscopy. Expression of BIP, ATF4, and CHOP were assessed by RT-PCR and Western blot. Testosterone/AR could not only initiate cell apoptosis but also induce the activation of eukaryotic initiation factor 2 alpha (eIF2α) cascades in INS-1 cells. Treatment of ER stress inhibitor or flutamide (AR inhibitor) could inhibit testosterone-induced cell apoptosis and CHOP expression. These results suggest that testosterone/AR pathway caused INS-1 cell apoptosis was at least in part through eIF2α/CHOP cascades.

Supplementation with a Leucine Pulse during Continuous Feeding Stimulates Translation Initiation and Suppresses Protein Degradation Pathways in Muscle of Neonatal Pigs

Leucine acts as a nutrient signal to stimulate protein synthesis in skeletal muscle. Limited evidence suggests that leucine may suppress protein degradation. Twenty piglets (8‐d‐old) received formula by orogastric tube for 24 h continuously (CON) or by bolus (BOL), i.e., every 4 h for 15 min. For the CON+LEU group, leucine was administered parenterally (800 μmol·kg−1·h−1) every 4 h for 1 h. Protein synthesis in skeletal muscle was greater in CON+LEU and BOL than CON. Ribosomal protein S6 kinase 1 and 4E‐binding protein 1 phosphorylation and active eukaryotic initiation factor (eIF) eIF4E·eIF4G complex formation were higher in muscle of CON+LEU and BOL than CON suggesting increased translation initiation. Eukaryotic elongation factor 2 and AMP‐activated protein kinase‐α phosphorylation was unaffected by treatment. The LC3‐II to total LC3 ratio was lower in CON+LEU and BOL than CON suggesting reduced autophagy‐lysosome activation. There were no differences between groups in indices of the ubiquitin‐proteasome pathway, i.e., Atrogin‐1 and MURF‐1 abundance and FOX03 phosphorylation. In conclusion, administration of a leucine pulse during continuous orogastric feeding increases protein synthesis by stimulating translation initiation and suppresses autophagy‐lysosome, but not the ubiquitin‐proteasome, degradation pathways in skeletal muscle of neonates. (NIH AR444474 and USDA/ARS 6250–51000‐055)

Lean Gain Is Enhanced by a Leucine Pulse during Long‐Term Continuous Feeding in Neonatal Pigs

Infants who are unable to maintain oral feeding can be nourished continuously by orogastric tube. We have previously shown in neonatal pigs that a leucine pulse administered during continuous feeding enhanced protein synthesis in skeletal muscle compared to continuous feeding alone for a 24 h period. Twenty‐three piglets (7‐d‐old) were fed continuously a milk replacement formula via orogastric tube for 21 d with an additional parenteral infusion of either leucine (800 μmol•kg−1•h−1, LEU) or alanine (ALA) for 1 h every 4 h. At 21 d, the body weight was higher and the weight gain greater in LEU than in ALA pigs. The lean gain, determined by dual‐energy X‐ray absorptiometry (DEXA), was higher and the fat gain lower in the LEU than ALA group. Longissimus dorsi (LD), gastrocnemius and soleus muscle and kidney weights were heavier in LEU than ALA with no effect of treatment on liver, small intestine and heart weights. Fractional protein synthesis rate and formation of the active eukaryotic initiation factor (eIF) eIF4E•eIF4G complex in LD were greater in the LEU than ALA group. In conclusion, administration of a leucine pulse during 21 d of continuous feeding decreases fat gain and increases lean gain by stimulating the translation initiation pathway leading to protein synthesis in skeletal muscle in neonates. (NIH AR444474 and USDA/ARS 6250–51000‐055)Grant Funding Source: NIH AR444474 and USDA/ARS 6250–51000‐055

Intermittent Bolus Feeding Enhances Lean Tissue Accretion by Increasing Muscle Amino Acid Transport and Protein Turnover Compared with Continuous Feeding in Neonates

Orogastric tube feeding is recommended in neonates unable to ingest food normally, and can be administered by intermittent bolus (INT) or continuous (CON) infusion. Recently, we showed that INT compared to CON feeding enhances lean tissue accretion by increasing muscle protein synthesis in neonates. To determine the mechanism for this response, neonatal pigs (n=6, 6‐d‐old) were fed the same diet in equivalent amounts continuously or intermittently (meal every 4h) for 21d. Insulin receptor and insulin receptor substrate 1 expression and protein kinase B phosphorylation in skeletal muscle were unaffected by feeding modality. Formation of the active eukaryotic initiation factor (eIF) 4E‐eIF4G complex and phosphorylation of ribosomal protein S6 kinase were higher and phosphorylation of eIF2α was lower in INT compared to CON fed pigs indicating increased translation initiation (P&lt;0.05). Sodium‐coupled neutral amino acid transporter 2, but not L‐type amino acid transporter 1, expression was higher in INT compared to CON pigs suggesting enhanced glutamine transport (P&lt;0.05). Abundance of the ubiquitin ligases, muscle RING finger‐1 and F‐box protein atrogin‐1/MAFbx, was higher for INT compared to CON pigs (P&lt;0.05). These results suggest that intermittent feeding enhances lean tissue accretion by increasing amino acid transport and protein turnover. (Supported by NIH AR444474 and USDA/ARS 6250–51000‐055)

Intermittent Bolus Feeding Increases Visceral Tissue Protein Synthesis More Than Continuous Feeding in Neonatal Pigs

Orogastric tube feeding, using either continuous or intermittent bolus delivery, is commonly used in infants unable to feed orally. To compare the impact of different feeding strategies on visceral tissue protein synthesis, neonatal pigs (5–7 day old) received a balanced formula orally either by intermittent bolus every 4 h (n=13) or continuously (n=9), or they were overnight fasted (n=6). Fractional protein synthesis and translational mechanisms in visceral tissues were examined at 0, 24, and 25.5 h. Plasma amino acid and insulin levels modestly increased in the continously fed group, whereas in the bolus meal group the pulsatile meal feeding pattern was mimicked. Compared to fasting, protein synthesis in the liver, pancreas, jejunum, and kidney increased in response to feeding in bolus and continuously fed pigs (P< 0.05), but the greatest increase occurred after a bolus meal (P< 0.001). Bolus meals, but not continuous feeds, increased the activation of eukaryotic initiation factor (eIF) 4E binding protein (4EBP1) and rp S6 kinase 1 (S6K1) (P< 0.05). Eukariotic elongation factor 2 (eEF2) and eIF2α phosphorylation were unaffected by both feeding modalities. These results suggest that intermittent bolus feeding increases protein synthesis in visceral tissues to a greater extent than continuous feeding by stimulating translation initiation. (Supported by NIH AR-44474 and AR-51563 and USDA/ARS 6250-51000-055).

HDAC2 Promotes eIF4E Sumoylation and Activates mRNA Translation Gene Specifically

Histone deacetylases (HDACs) are best known as transcription corepressors through deacetylating histone tails. Here we show that HDAC2 is also involved in cap-dependent mRNA translation by promoting sumoylation of eukaryotic translation initiation factor 4E (eIF4E), which is independent of its deacetylase activity. By stimulating eIF4E sumoylation, HDAC2 induces the formation of the active eukaryotic initiation factor 4F (eIF4F) complex and induces the protein synthesis of a subset of eIF4E-responsive genes that are essential for cell proliferation and preventing apoptosis. These data demonstrate that HDAC2 has an unexpected sumoylation-promoting activity and regulates cap-dependent mRNA translation.

Intermittent Bolus Feeding Has a Greater Stimulatory Effect on Protein Synthesis in Skeletal Muscle than Continuous Feeding in Neonatal Pigs

Feeding by orogastric tube, using either continuous or intermittent bolus delivery, is common for infants for whom normal feeding is contraindicated. To determine the impact of different feeding modalities on muscle protein anabolism, neonatal pigs (5–7 day old) received a balanced enteral formula either by intermittent bolus (B, n=7) every four hours or were continuously fed (C, n=5). Fractional protein synthesis rates and translational control mechanisms in skeletal muscle were examined at 0, 24 and 25.5 hours. Plasma amino acid and insulin levels were modestly increased in the continuously fed group and in the bolus group mimicked the pulsatile pattern of meal feeding. Feeding increased protein synthesis by 53% in the continuous group, by 63% in the bolus group before a meal, and by 126 % after a bolus feed. The activation of eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), rp S6 kinase 1 (S6K1), protein kinase B (PKB), dissociation of 4EBP1‐eIF4E complex, and formation of the active eIF4E‐eIF4G complex were unaffected by continuous feeding but were increased more than 10 fold after a bolus meal. These results suggest that intermittent bolus feeding increases muscle protein synthesis to a greater extent than continuous feeding by stimulating translation initiation. (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

Oral Leucine Enhances Myocardial Protein Synthesis in Rats Acutely Administered Ethanol ,

Acute alcohol ingestion induces an inhibition of myocardial protein synthesis by impairing mRNA translation initiation. Elevating plasma leucine (Leu) concentrations via oral gavage stimulates mRNA translation initiation in several tissues, although the effect in heart has not been well defined. The experiments described herein were designed to test the effects of a gavage solution containing Leu on protein synthesis and potential mechanisms important in accelerating mRNA translation initiation in cardiac muscle of rats given ethanol acutely to mimic “binge” dinking. Gavage with Leu stimulated protein synthesis and enhanced the assembly of the active eukaryotic initiation factor (eIF)4G·eIF4E complex. Increased assembly of the active eIF4G·eIF4E complex was associated with a 130% rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly (∼30%) of inactive eIF4E-binding protein1 (4EBP1)·eIF4E complex in rats-administered ethanol. The reduced assembly of the 4EBP1·eIF4E complex was associated with an increase in phosphorylation of 4EBP1 in the hyperphosphorylated γ-form following Leu gavage. Phosphorylation of mammalian target of rapamycin on Ser2448, an upstream regulator of phosphorylation of 4EBP1, was elevated following Leu gavage. Neither the phosphorylation of 70-kDa ribosomal protein S6 kinase on Thr389 nor eIF4E phosphorylation was increased following Leu gavage under any condition. Leu gavage accelerates myocardial protein synthesis following acute ethanol intoxication by enhancing eIF4G·eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4EBP1 with eIF4E.

Acute Oral Leucine Administration Stimulates Protein Synthesis during Chronic Sepsis through Enhanced Association of Eukaryotic Initiation Factor 4G with Eukaryotic Initiation Factor 4E in Rats ,

Sepsis induces the loss of muscle proteins by impairing skeletal muscle protein synthesis through an inhibition of messenger RNA (mRNA) translation initiation. Amino acids and Leu (Leu) in particular stimulate mRNA translation initiation. The experiments were designed to test the effects of Leu on potential signal transduction pathways that may be important in accelerating mRNA translation initiation in skeletal muscle of rats with chronic (5–6 d) septic intra-abdominal abscess. Gastrocnemius from male Sprague Dawley rats gavaged with Leu or water were sampled 5–6 d following development of an intra-abdominal sterile or septic abscess. Gavage with Leu stimulated protein synthesis and enhanced the assembly of the active eukaryotic initiation factor (eIF)4G-eIF4E complex. Increased assembly of the active eIF4G-eIF4E complex was associated with a robust rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly of inactive eIF4E binding protein-1 (4E-BP1)-eIF4E complex in both sterile inflammatory and septic rats. The reduced assembly of 4E-BP1-eIF4E complex was associated with an increase in phosphorylation of 4E-BP1 in the γ-form following Leu gavage. Phosphorylation of 70-kDa ribosomal protein S6 kinase on Thr389 was also increased following Leu gavage, as well as the phosphorylation of mammalian target of rapamycin on Ser2448 or Ser2481. In contrast, phosphorylation of protein kinase B (PKB) on Thr308 or Ser473 was not augmented following Leu gavage in septic rats. We conclude that Leu stimulates a PKB-independent signal pathway elevating the eIF4G-eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E in skeletal muscle during sepsis.

Rapamycin Limits Formation of Active Eukaryotic Initiation Factor 4F Complex Following Meal Feeding in Rat Hearts ,

Feeding promotes protein synthesis in cardiac muscle through a stimulation of the messenger RNA translation initiation phase of protein synthesis by enhancing assembly of active eukaryotic initiation factor (eIF)4F complex. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in increasing formation of active eIF4G-eIF4E complex during meal feeding. Hearts from male Sprague-Dawley rats fed a meal consisting of rat nonpurified diet were sampled prior to and 3 h following the meal in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1. Rapamycin prevented the meal feeding-induced stimulation of myocardial protein synthesis. Inhibition of mTOR with rapamycin decreased the association of rapamycin-associated TOR protein with mTOR and prevented the feeding-induced assembly of eIF4G-eIF4E complex. In contrast, the abundance of eIF4E binding protein-1 (4E-BP1)-eIF4E complex was unaffected by either meal feeding or rapamycin. Pretreatment with rapamycin completely prevented the feeding-induced phosphorylation of eIF4G(Ser1108), whereas the inhibitor only partially attenuated meal feeding-induced 70-kDa ribosomal protein S6 kinase1(Thr389) phosphorylation and extent of 4E-BP1 in the γ-form. Meal feeding-induced phosphorylation of protein kinase B on either Ser473 or Thr308 was unaffected by rapamycin. These findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs by a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex.

Sex-dependent differences in the regulation of myocardial protein synthesis following long-term ethanol consumption

Chronic heavy alcohol consumption alters cardiac structure and function. Controversies remain as to whether hearts from females respond to the chronic ethanol intake in a manner analogous to males. In particular, sex differences in the myocardial response to chronic alcohol consumption remain unresolved at the molecular level. The purpose of the present set of experiments was to determine whether alterations in cardiac structure and protein metabolism show sexual dimorphism following chronic alcohol consumption for 26 wk. In control animals, hearts from female rats showed lowered heart weights and had thinner ventricular walls compared with males. The smaller heart size was associated with a lower protein content that occurred in part from a reduced rate of protein synthesis. Chronic alcohol consumption in males, but not in females, caused a thinning of the ventricular wall and intraventricular septum, as assessed by echocardiography, correlating with the loss of heart mass. The alterations in cardiac size occurred, in part, through a lowering of the protein content secondary to a diminished rate of protein synthesis. The decreased rate of protein synthesis appeared related to a reduced assembly of active eukaryotic initiation factor (eIF)4G.eIF4E complex secondary to both a diminished phosphorylation of eIF4G and increased formation of inactive 4Ebinding protein (4EBP1).eIF4E complex. The latter effects occurred as a result of decreased phosphorylation of 4EBP1. None of these ethanol-induced alterations in hearts from males were observed in hearts from females. These data suggest that chronic alcohol-induced impairments in myocardial protein synthesis results, in part, from marked decreases in eIF4E.eIF4G complex formation in males. The failure of female rats consuming ethanol to show structural changes appears related to the inability of ethanol to affect the regulation protein synthesis to the same extent as their male counterparts.

Alcohol and Indinavir Adversely Affect Protein Synthesis and Phosphorylation of MAPK and mTOR Signaling Pathways in C2C12 Myocytes

Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S]methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

Arginine/Arginase NO NO NO

Arginine is a semi-essential amino acid that plays a critical role in several metabolic pathways. The best known of these is as the immediate precursor of urea in the liver and to a lesser extent in the intestine.1 The enzyme responsible for the cleavage of arginine to produce urea is arginase, which is the only enzyme in the urea cycle that comes in two forms, arginase I, a cytosolic enzyme, and arginase II, a mitochondrial enzyme. These enzymes exist not only in the liver and intestine where urea is generated but are also widely distributed in the body. Ornithine, the other product of the cleavage of arginine by arginase, is a precursor of proline and polyamines such as spermine, spermidine, and putrescine. Arginine is also the precursor of nitric oxide (NO). See page 365 In an important article authored by Teupser and colleagues,2 arginase in macrophages has been identified as a candidate gene influencing atheroresponsiveness. They created and studied two strains of rabbits with genetically determined high (HAR) and low (LAR) atherosclerosis susceptibility. Subtractive suppression hybridization was used to screen for genes that were more highly expressed in macrophages from LAR rabbits. Among the 42 clones identified were four differentially expressed genes with potential relevance to atherogenesis. These were OC2 protein, fibronectin, ferritin H-chain, and arginase I. Despite their identification in the screen, the first three genes were not differentially expressed in macrophages between HAR and LAR animals when checked by quantitative RT-PCR. In contrast, arginase I was more highly expressed in LAR than HAR macrophages, and its expression level was correlated with higher arginase enzymatic activity. They further identified a length polymorphism of the mRNA for arginase I that was attributable to the presence (long variant) or absence (short variant) of a 413-bp C repeat in the …

IGF-I activates the eIF4F system in cardiac muscle in vivo

IGF-I acutely stimulates protein synthesis in cardiac muscle through acceleration of mRNA translation. In the present study, we examined the regulatory signaling pathways and translation protein factors that potentially contribute to the myocardial responsiveness of protein synthesis to IGF-I in vivo. IGF-I was injected IV into rats and 20 min later the hearts were excised and homogenized for assay of regulatory proteins. IGF-I increased assembly of the translationally active eukaryotic initiation factor (eIF)4G.eIF4E complex. The increased assembly of eIF4G.eIF4E was associated with an enhanced eIF4G phosphorylation and increased availability of eIF4E. Increased availability of eIF4E occurred as a consequence of diminished abundance of the inactive 4E-BP1.eIF4E complex following IGF-I. The assembly of the 4E-BP1.eIF4E complex appeared to be decreased through an IGF-I-induced phosphorylation of 4E-BP1. IGF-I also caused an increase in the phosphorylation of S6K1. Activation of the potential upstream regulators of 4E-BP1 and S6K1 phosphorylation via PKB and mTOR was also observed. In contrast, there was no effect of IGF-I on phosphorylation of elongation factor (eFE)2. The results suggest the major impact of IGF-I in cardiac muscle occurred via stimulation of translation initiation rather than elongation. Furthermore, the results are consistent with a role for assembly of active eIF4G.eIF4E complex and activation of S6K1 in mediating the stimulation of mRNA translation initiation by IGF-I through a PKB/mTOR signaling pathway.