Eukaryotic Initiation Factor 4E-binding Protein Research Articles

The Bi-Steric Inhibitor RMC-5552 Reduces mTORC1 Signaling and Growth in Lymphangioleiomyomatosis.

Mutations in the Tuberous Sclerosis Complex (TSC) genes result in the hyperactivation of the mechanistic/mammalian target of rapamycin 1 (mTORC1) growth pathway in mesenchymal pulmonary cells. Rapamycin (SirolimusTM), a naturally occurring macrolide, is the only therapeutic approved for women with lymphangioleiomyomatosis (LAM), a progressive, destructive lung disease caused by TSC gene mutations and mTORC1 hyperactivation. However, on cessation of the drug, lung function decline continues. We demonstrated here that pulmonary LAM cancer stem-like cells (SLS) most highly expressed the eukaryotic translation initiation factor 4E (eIF4E)-dependent translation initiation genes. We also showed that the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) gene has the lowest expression in these cells, indicating that the 4E-BP1/eIF4E ratio in LAM SLS cells favors unrestrained eIF4E oncogenic mRNA translation. The bi-steric mTORC1-selective compound RMC-5552 prevented growth of LAM-associated fibroblasts (LAFs) and phosphorylation of proteins in the ribosomal protein S6K1/ribosomal protein S6 (S6K1/S6) and 4E-BP1/eIF4E translation mTORC1-driven pathways, whereas rapamycin only blocked the S6K/S6 axis. Rapamycin inhibition of LAF growth was rapidly reversed, but RMC-5552 inhibition was more durable. RMC-5552, through its potential to eradicate LAM cancer SLS cells, may have therapeutic benefit in LAM and other diseases with mTORC1 hyperactivity.

Regulatory-Associated Protein of mTOR-Mediated Signaling: A Nexus Between Tumorigenesis and Disease

RAPTOR (regulatory-associated protein of mTOR) is a pivotal component of the mammalian target of rapamycin complex 1 (mTORC1), playing a central role in regulating cell growth, metabolism and stress responses. As a scaffold protein, RAPTOR recruits key substrates such as eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 kinase (S6K), facilitating their phosphorylation by mTORC1, which in turn drives protein synthesis, lipid metabolism and cellular proliferation. Its regulatory function becomes especially crucial under conditions of nutrient deprivation or stress, where it enhances the stability of the mTORC1 complex, allowing cells to adapt to fluctuating environmental cues. The hyperactivation of mTORC1, largely mediated by RAPTOR, is frequently observed in various cancers, contributing to uncontrolled cell proliferation and tumorigenesis. Moreover, RAPTOR’s modulation of immune responses and metabolic pathways extends its influence beyond oncogenesis, impacting inflammatory diseases and metabolic disorders. This review meticulously elucidates RAPTOR’s structure, post-translational modifications as well as its indispensable role within the mTORC1 complex, emphasizing its regulatory functions in cellular growth, metabolic adaptation, immune response and disease pathology including oncogenesis. Furthermore, it explores emergent therapeutic avenues targeting RAPTOR-mediated mTORC1 signaling, underscoring their potential to revolutionize cancer treatment and the management of related pathophysiological conditions.

Causal Relationships of Circulating Inflammatory Proteins and Portal Vein Thrombosis: A Mendelian Randomization Study.

Portal vein thrombosis (PVT) is commonly encountered in patients with cirrhosis, challenging our understanding of its development, particularly the ambiguous contribution of inflammation. This study utilized Mendelian randomization (MR) to explore the causal impact of circulating inflammatory markers on PVT.Employing a two-sample MR framework, we merged genome-wide association study (GWAS) meta-analysis findings of 91 inflammation-associated proteins with independent PVT data from the FinnGen consortium's R10 release. A replication analysis was performed using a distinct GWAS dataset from the UK Biobank. Inverse variance weighting, MR-Egger regression, weighted median estimator, and Mendelian Randomization Pleiotropy RESidual Sum and Outlier were used for analysis, supplemented by multivariable MR (MVMR) to adjust for cirrhosis effects.Findings indicate a significant inverse association between the genetically inferred concentration of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and PVT risk, evidenced by an odds ratio (OR) of 0.37 (95% confidence interval [CI]: 0.21-0.67; p = 9.2 × 10-4; adjusted for multiple testing p = 0.084). This association was corroborated in the replication phase (OR = 0.39, 95% CI: 0.17-0.93; p = 0.03) and through MVMR analysis (OR = 0.34, 95% CI: 0.15-0.79; p = 0.012). Sensitivity analyses disclosed no evidence of heterogeneity or pleiotropy.Our investigation emphasizes the 4E-BP1 as a protective factor against PVT, underscoring its potential relevance in understanding PVT pathogenesis and its implications for diagnosis and therapy.

The Spectrum of Renal "TFEopathies": Flipping the mTOR Switch in Renal Tumorigenesis.

The mammalian target of Rapamycin complex 1 (mTORC1) is a serine/threonine kinase that couples nutrient and growth factor signaling to the cellular control of metabolism and plays a fundamental role in aberrant proliferation in cancer. mTORC1 has previously been considered an "on/off" switch, capable of phosphorylating the entire pool of its substrates when activated. However, recent studies have indicated that mTORC1 may be active toward its canonical substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase (S6K), involved in mRNA translation and protein synthesis, and inactive toward TFEB and TFE3, transcription factors involved in the regulation of lysosome biogenesis, in several pathological contexts. Among these conditions are Birt-Hogg-Dubé syndrome (BHD) and, recently, tuberous sclerosis complex (TSC). Furthermore, increased TFEB and TFE3 nuclear localization in these syndromes, and in translocation renal cell carcinomas (tRCC), drives mTORC1 activity toward the canonical substrates, through the transcriptional activation of the Rag GTPases, thereby positioning TFEB and TFE3 upstream of mTORC1 activity toward 4EBP1 and S6K. The expanding importance of TFEB and TFE3 in the pathogenesis of these renal diseases warrants a novel clinical grouping that we term "TFEopathies." Currently, there are no therapeutic options directly targeting TFEB and TFE3, which represents a challenging and critically required avenue for cancer research.

Tumoral EIF4EBP1 regulates the crosstalk between tumor-associated macrophages and tumor cells in MRTK

Malignant renal rhabdoid tumor (MRTK) is an aggressive and rare malignancy primarily affecting infants and young children. The intricate interactions within the Tumor Microenvironment (TME) are crucial in shaping MRTK's progression. This study elucidates the significance of tumor-associated macrophages(TAMs) within this milieu and their interplay with eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) in tumor cells, collectively contributing to MRTK's malignant advancement. Through comprehensive analysis of clinical samples and the TARGET database, EIF4EBP1 emerges as a central macrophage-associated gene with robust prognostic implications. Elevated EIF4EBP1 expression correlates with poor prognosis and heightened infiltration of TAMs. Functional validation demonstrates that EIF4EBP1 knockdown in G401 cells significantly attenuates self-proliferation, migration, and invasion. Moreover, EIF4EBP1 regulates macrophage recruitment and M2 polarization through the ERK/P38 MAPK-MIF axis. Notably, M2 macrophages reciprocally foster the malignant behavior of MRTK tumor cells. This study unveils the pivotal role of EIF4EBP1 in propelling MRTK's malignant progression, unraveling a complex regulatory network involving EIF4EBP1 and TAMs. These findings underscore EIF4EBP1 as a promising biomarker and highlight its therapeutic potential in MRTK management.

Genetically predicted 91 circulating inflammatory proteins in relation to risk of urological malignancies: a Mendelian randomization study.

Urological malignancies, including kidney, bladder, and prostate cancer, are major health concerns worldwide. Inflammation has been implicated in the pathogenesis of these cancers, and circulating inflammatory proteins may play a role in their development. However, the causal relationship between specific plasma proteins and urological malignancies remains unclear. We performed a two-sample Mendelian randomization (MR) analysis using summary statistics from genome-wide association studies (GWAS). Instrumental variables representing genetic variants associated with circulating inflammatory proteins were used to infer causality on the risk of kidney, bladder, and prostate cancer. Four MR methods were utilized to provide robust effect estimates. Our analysis identified several plasma proteins associated with a lower risk of kidney and bladder cancer, including Eukaryotic translation initiation factor 4E-binding protein 1, Caspase 8, Natural killer cell receptor 2B4, and Tumor necrosis factor ligand superfamily member 12. However, after adjusting for multiple testing, these associations did not remain statistically significant. For prostate cancer, CUB domain-containing protein 1 and Interleukin-10 receptor subunit beta were found to be protective, while Glial cell line-derived neurotrophic factor and SIR2-like protein 2 were identified as risk factors. After FDR adjustment, none of the inflammatory proteins were found to be significantly associated with a lower risk of prostate cancer. Our findings suggest that certain plasma proteins may be involved in the development of urological malignancies. Mendelian randomization provides a useful framework for investigating causal relationships between inflammatory proteins and urological cancers, offering potential insights into their underlying biology and therapeutic targets.

Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

The histone methyltransferase SUV420H2 regulates brown and beige adipocyte thermogenesis.

Activation of brown adipose tissue (BAT) thermogenesis increases energy expenditure and alleviates obesity. Here we discover that histone methyltransferase suppressor of variegation 4-20 homolog 2 (Suv420h2) expression parallels that of Ucp1 in brown and beige adipocytes and that Suv420h2 knockdown significantly reduces - whereas Suv420h2 overexpression significantly increases - Ucp1 levels in brown adipocytes. Suv420h2 knockout (H2KO) mice exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. In contrast, mice with specific overexpression of Suv420h2 in adipocytes display enhanced cold-induced thermogenesis and are resistant to diet-induced obesity. Further study shows that Suv420h2 catalyzes H4K20 trimethylation at eukaryotic translation initiation factor 4E-binding protein 1 (4e-bp1) promoter, leading to downregulated expression of 4e-bp1, a negative regulator of the translation initiation complex. This in turn upregulates PGC1α protein levels, and this upregulation is associated with increased expression of thermogenic program. We conclude that Suv420h2 is a key regulator of brown/beige adipocyte development and thermogenesis.

Analysis of the stimulative effect of arginine on translation initiation of protein synthesis in skeletal muscle

Introduction: Amino acids are not only important precursors for the synthesis of proteins, but also participate in the regulation of major metabolic pathways. Specific amino acids stimulate protein synthesis. Previous studies have shown that arginine (Arg) stimulates protein synthesis at the level of translation initiation through the activation of the mechanistic target of rapamycin complex 1 (mTORC1) in cell culture system; however, little is known about the effect of Arg on protein synthesis in vivo. Here, we evaluated whether Arg stimulates protein synthesis at the level of translation initiation and compared the effects of Arg on translation initiation with those of leucine (Leu), which is well-studied the detail of its stimulative mechanism, in mouse skeletal muscle. Methods: Eighteen hour-fasted mice were intraperitoneally injected with saline or 4.6 mmol/ kg/ body weight of Arg or Leu and subsequently sacrificed and excised gastrocnemius muscles at 1 h after injection. In addition, we used mouse-derived C2C12 myotubes treated with Arg to examine the mechanism by which Arg stimulates translation initiation in mouse skeletal muscle. The cells were preincubated with several kinds of inhibitors for appropriate time and then treated with Arg or Leu. Using homogenate of skeletal muscle and cell lysate, the phosphorylation states of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1), key regulatory factors involved in the translation initiation were measured by immunoblotting as indicators of translation initiation activity. Results: Intraperitoneal injection of either Arg or Leu significantly increased S6K1 phosphorylation compared to the saline-injection, but the increase in 4E-BP1 phosphorylation was observed in Leu-injected mice not in Arg-injected mice. These results suggest that Arg can stimulate translation initiation in skeletal muscle of fasted mice. In C2C12 myotubes, as with Leu, Arg increased both 4E-BP1 and S6K phosphorylation, and these increases were abolished by the treatment of rapamycin, a mTORC1 inhibitor. These data indicate that Arg directly activates mTORC1 pathway as well as Leu. In addition, the treatment of a phosphoinositide 3-kinase (PI3K) inhibitor and a AKT inhibitor completely cancelled Arg-dependent 4E-BP1 and S6K1 phosphorylation; however, the inhibition of Leu-induced 4E-BP1 and S6K1 phosphorylation by these inhibitors were in partial. Furthermore, the inhibitor of the G protein‐coupled receptor family C, group 6, subtype A (GPRC6a), which has been shown to associate with regulation of PI3K/AKT pathway and mTORC1 activation, completely suppressed the increases of 4E-BP1 and S6K1 phosphorylation by Arg. Conclusion:Our data indicate that Arg has the ability to activate translation initiation in mouse skeletal muscle. In addition, the results of our experiments using skeletal muscle cells support the idea that Arg stimulates translation initiation via GPRC6a/PI3K/AKT/mTORC1 pathway in mouse skeletal muscle. Unlike the effects of Arg, it is likely that besides the PI3K/AKT pathway, Leu activates another signaling pathways upstream of mTORC1 to stimulate translation initiation in skeletal muscle. This research was funded by JSPS KAKENHI, grant number 18K05501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E–binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at −80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

Bone characteristics, pre-caecal phytate degradation, mineral digestibility and tissue expression were marginally affected by zinc level and source in phytase-supplemented diets in 21-day-old broiler chickens

ABSTRACT 1. This study determined the effect of dietary Zn concentration and source in phytase-supplemented diets on bone mineralisation, gastrointestinal phytate breakdown, mRNA-level gene expression (in jejunum, liver and Pectoralis major muscle) and growth performance in broiler chickens. 2. Male Cobb 500 broilers were housed in floor pens (d 0-d 21) to test seven treatments with six replicate pens (12 birds per pen). Diets were arranged in a 2 × 3 + 1-factorial arrangement. The experimental factors were Zn source (Zn-oxide (ZnO) or Zn-glycinate (ZnGly) and Zn supplementation level (10, 30 or 50 mg/kg of diet). A maize-soybean meal-based diet without supplementation and formulated to contain 28 mg Zn/kg (analysed to be 35 mg Zn/kg), served as a control. 3. Zinc source and level did not influence (p > 0.05) bone ash concentration and quantity or mineral concentrations in bone ash. Tibia thickness was greater in the treatment ZnO10 than in the treatments ZnO30 and ZnGly50 (Zn level × Zn source: p = 0.036), but width and breaking strength were not affected. 4. Pre-caecal P digestibility and concentrations of phytate breakdown products in the ileum, except for InsP5, were not affected by Zn source or level. Only the expression of EIF4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1) and FBXO32 (F-box only protein 32) in Pectoralis major muscle was affected by source, where expression was increased in ZnO compared to ZnGly diets (p < 0.05). 5. In conclusion, Zn level and source did not affect gastrointestinal phytate degradation and bone mineralisation in phytase-supplemented diets. The intrinsic Zn concentration appeared to be sufficient for maximum bone Zn deposition under the conditions of the present study but requires validation in longer-term trials.

FKBP38 suppresses endometrial cancer cell proliferation and metastasis by inhibiting the mTOR pathway

Endometrial cancer (EC) is a common gynecological malignancy, and advanced-stage or recurrent EC is associated with a high mortality rate owing to the ineffectiveness of currently available treatments. FK506-binding protein 38 (FKBP38) is a member of the immunophilin family and inhibits melanoma and breast cancer cell metastasis. However, the functions of FKBP38 and its potential mechanism in EC remain unclear. Herein, we analyzed the expression levels of FKBP38 in EC cells and found that the FKBP38 expression was high in Ishikawa cells, and low in AN3CA cells, traditionally considered a low grade and a high grade cell line, respectively, in pathology classification. Moreover, FKBP38 inhibited cell proliferation, migration and invasion in EC cells, FKBP38 knockdown significantly promoted tumor growth of Ishikawa cells in a subcutaneous xenograft model and increased the number of lung metastases of Hec-1-A cells in a metastatic mouse model. Furthermore, FKBP38 suppressed several target proteins of epithelial-to-mesenchymal transition (EMT) and reduced the phosphorylation of ribosomal S6 protein (S6), eukaryotic initiation factor 4E-binding protein 1 (4EBP-1), indicating the potent inhibition of the mammalian target of rapamycin (mTOR) pathway. Meanwhile, the inhibition of mTOR neutralized the elevation of EC cell proliferation, migration and invasion after FKBP38 knockdown. In summary, FKBP38 would exert a tumor-suppressing role by modulating the mTOR pathway. Our results indicate that FKBP38 may be considered as a factor of EC metastasis and a new target for EC therapeutic intervention.

Dietary glycine supplementation activates mechanistic target of rapamycin signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

MTOR inhibition modulates apoptosis and oxidative stress in hindlimb ischemia/reperfusion injury

Purpose: Ischemia/reperfusion (I/R)-induced injuries represent serious clinical events regarding profound target organ destructions followed by remote organ complications due to the loss of oxidant/antioxidant balance and apoptosis. Recent studies examining the mammalian target of rapamycin (mTOR) during I/R injury in different organs have remained a matter of debate. The current study aimed to explore further the protective and underlying antiapoptotic and antioxidant mechanisms of mammalian target of rapamycin (mTOR) inhibition in hindlimb (HL) schemia/reperfusion (I/R)injury. &#x0D; Materials and Methods: Occlusion of bilateral hindlimbs for 4 h with tourniquets was carried out under anesthesia to induce I/R for 4 h in rats. Rapamycin (1 mg/kg) or saline (4 mL/kg) was injected intraperitoneally 1 h before reperfusion. Gastrocnemius muscle, kidney, and blood were collected at the end of the experiments for analysis. Muscle and kidney damages were evaluated by measuring protein expression and/or phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 (rpS6), B-cell lymphoma 2 (Bcl-2), caspase-3, and Bcl-2-associated X protein (Bax) with NADPH oxidase level and total antioxidant capacity in tissues or sera. &#x0D; Results: I/R-induced organ damages were demonstrated by enhanced phosphorylation and/or expression of rpS6, 4EBP1, caspase-3, and Bax with a significant reduction in Bcl-2 accompanied by a decreased total antioxidant capacity and increased level of NADPH oxidase. Administration of rapamycin, an inhibitor mTOR, protected against I/R-mediated injuries. &#x0D; Conclusion: Our findings suggest that the activation of mTOR signaling plays a crucial role in HL I/R-triggered organ damages presumably through the activation of apoptosis as a result of oxidant/antioxidant imbalance.

Effects of Low-Load, High-Repetition Resistance Training on Maximum Muscle Strength and Muscle Damage in Elite Weightlifters: A Preliminary Study

This study aimed to assess the impact of different resistance training (RT) loads and repetition on muscle damage, intramuscular anabolic signaling, and maximal muscle strength (MMS) in weightlifters. Eighteen male weightlifters were randomly assigned to 8 weeks of supervised RT regimes: high-load, low-repetition (HL), low-load, high-repetition (LH), and combination of HL and LH (COMBI). All groups exhibited a significant increase in skeletal muscle mass (SMM) and growth hormone levels, which ultimately contributed to improvement in MMS as indicated by 1-repetition maximum in the back squat and back muscle strength. Notably, while there were no significant changes in the mTOR protein, the phosphorylation of phosphorylation of p70 ribosomal protein S6 kinase 1 (p70S6K1), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic elongation factor 2 (eEF2), which are involved in muscle cell growth, was significantly affected by the different training regimens. More importantly, LH-RT led to a significant reduction in muscle damage markers, creatine kinase (CK) and lactate dehydrogenase (LDH), suggesting reduced recovery time and fatigue. Our results demonstrated that the LH-RT paradigm could be a viable alternative for weightlifters to enhance MMS and muscle hypertrophy similar to HL-RT, while reducing RT-induced muscle damage, ultimately contributing to the enhancement of exercise performance.

4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling.

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

4EBP2-regulated protein translation has a critical role in high-fat diet–induced insulin resistance in hepatocytes

A high-fat diet (HFD) plays a critical role in hepatocyte insulin resistance. Numerous models and factors have been proposed to elucidate the mechanism of palmitic acid (PA)-induced insulin resistance. However, proteomic studies of insulin resistance by HFD stimulation are usually performed under insulin conditions, leading to an unclear understanding of how a HFD alone affects hepatocytes. Here, we mapped the phosphorylation rewiring events in PA-stimulated HepG2 cells and found PA decreased the phosphorylation level of the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2) at S65/T70. Further experiments identified 4EBP2 as a key node of insulin resistance in either HFD mice or PA-treated cells. Reduced 4EBP2 levels increased glucose uptake and insulin sensitivity, whereas the 4EBP2_S65A/T70A mutation exacerbated PA-induced insulin resistance. Additionally, the nascent proteome revealed many glycolysis-related proteins translationally regulated by 4EBP2 such as hexokinase-2, pyruvate kinase PKM, TBC1 domain family member 4, and glucose-6-phosphate 1-dehydrogenase. In summary, we report the critical role of 4EBP2 in regulating HFD-stimulated insulin resistance in hepatocytes.

Dietary melatonin positively impacts the immune system of crayfish, Cherax destructor, as revealed by comparative proteomics analysis

Melatonin, an indoleamine with various biological activities, is being used increasingly in the aquaculture industry for its broad immune effects. Cherax destructor is an emerging economically cultured crayfish that faces many problems in the breeding process. Previous work found that dietary melatonin has positive effects on the growth and immunity of C. destructor, but the specific mechanism involved remained unclear. In this study, proteomics was used to determine the mechanism of action of melatonin in C. destructor. Results showed that dietary melatonin resulted in decreased levels of hydrogen peroxide, alanine aminotransferase, and aspartate aminotransferase, but increased levels of glutathione peroxidase, acid phosphatase, and glutathione S-transferases. In total, 608 proteins were differentially expressed (418 upregulated and 190 downregulated), and were enriched in three main categories: innate immunity (B cell receptor signaling pathway and natural killer cell-mediated cytotoxicity), glucose metabolism (pentose phosphate pathway, pentose and glucuronate interconversions, and propionate metabolism), and amino acid metabolism (valine, leucine, and isoleucine degradation, and cysteine and methionine metabolism). In addition, dietary melatonin was also involved in the regulation of the mTOR signaling pathway, and upregulated the expression of genes encoding key factors, such as Ras-related GTP-binding protein A/B, eukaryotic initiation factor 4E, eukaryotic initiation factor 4E-binding protein, and p70 ribosomal S6 kinase. Overall, this study demonstrates the role of melatonin in the physiological regulation of C. destructor, laying the foundation for the development of melatonin as a feed additive in the aquaculture of this species.

Effects of dietary carbohydrate/lipid ratios on growth, body composition, amylase activity, oxidative status, and mTOR/autophagy pathway in juvenile clam, Sinonovacula constricta

The nutritional requirements and metabolism of bivalves remain largely unknown. In this study, we investigated the effects of dietary carbohydrate/lipid ratios on growth performance, body composition, digestive enzyme activities, oxidative status, and transcriptome profile in juvenile razor clam Sinonovacula constricta. Five microcapsule diets with varying levels of carbohydrate (30–45%) and lipid (2–15%) were prepared and designated as C45L2 (45% carbohydrate and 2% lipid), C41L5, C36L8, C33L11, and C30L15 groups. Juvenile S. constricta with an initial shell length of 4.9 mm were fed with these microcapsule diets for 2 weeks. The results showed that the clam in the C33L11 group exhibited the fastest growth rate. The body protein content decreased with the decreasing dietary carbohydrate/lipid ratio. The clam in the C33L11 group showed significantly higher glycogen content compared to the C45L2, C41L5, or C30L5 group. The amylase activity of the clam in the C45L2 and C41L5 group was significantly higher than that in the C36L8, C33L11, and C30L15 group. The clam in the C33L11 group showed significantly higher catalase and superoxide dismutase activities compared to other microcapsule groups. The malondialdehyde level of the clam in the C30L15 group was significantly higher than that in the C45L2, C41L5, and C33L11 group. Transcriptome analysis showed that the autophagy pathway in the clam fed C41L5 was upregulated compared with those fed C33L11. Meanwhile, the protein levels of microtubule-associated protein 1 A/1B-light chain 3 membrane type (LC3-II) in the C41L5 group were also significantly higher than those in the C33L11 group. Moreover, the clam in the C41L5 group exhibited increased phosphorylation levels of the eukaryotic translation initiation factor 4E-binding protein (4E-BP) and decreased phosphorylation levels of the ribosomal protein S6 kinase beta-1 (S6K). Our results revealed that the optimal dietary carbohydrate/lipid ratio for juvenile S. constricta was approximately 3.01. The high-carbohydrate/low-lipid diet may repress the growth of clam by inhibiting mammalian target of rapamycin complex 1 (mTORC1) and subsequently activating autophagy, while the poor growth performance in the low-carbohydrate/high-lipid diet group may be associated with high lipid peroxidation levels in juvenile S. constricta.

Abstract P3194: Redd1 Is Required For Glucose Metabolism And Insulin Sensitivity In Cardiomyocytes

Type II diabetes (TIID) is characterized by insulin resistance (IR) and doubles a patient’s risk for cardiovascular disease (CVD). The pathogenesis of CVD in patients with TIID, or diabetic cardiomyopathy (DC), is unclear. Regulated in development and DNA damage (REDD)1 is a negative regulator of mammalian target of rapamycin complex (mTORC)1. Notably, deletion of REDD1 leads to systemic IR in mice. REDD1 is, therefore, necessary for insulin sensitivity, however the mechanisms required remain unclear. We hypothesized that REDD1 is required for glucose oxidation and, therefore, insulin sensitivity in cardiomyocytes. To examine the role of REDD1 in cardiomyocyte glucose metabolism and energetics, we cultured wild type (WT) AC16 cardiomyocytes or those with REDD1 deletion with 5.5 mM glucose and examined cellular respiration. The data demonstrate compromised respiration, as indicated by reduced maximal respiratory capacity in REDD1-null versus WT cells (1.5-fold±0.214). Importantly, western blotting confirmed the absence of REDD1 in cells with REDD1 deletion, as well as increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (p4E-BP)1 (Thr37/46) (1.3-fold±0.085), a direct target of mTORC1. Thus, REDD1 inhibits mTORC1 signaling and enhances glucose-driven respiration in cardiomyocytes. To examine the mechanisms by which REDD1 supports glucose utilization, we examined levels of phosphorylated pyruvate dehydrogenase (pPDH) (Ser293). Our data show that pPDH is increased in REDD1-null cells versus WT controls (1.2-fold±0.054), as well as in hearts from REDD1-deficient mice (1.2-fold±0.013), suggesting reduced glucose oxidation. We also observed enhanced extracellular acidification, an indirect measure of glycolysis, in our REDD1-null cells versus WT controls (1.7-fold±0.136). Pyruvate dehydrogenase kinases (PDKs) phosphorylate PDH. In our REDD1-null cells, we observe a significant increase in PDK4 mRNA versus WT controls (8-fold±0.593), as assessed by qPCR and consistent with the diabetic heart. Together, these data suggest that REDD1 inhibits mTORC1 activity and the expression of PDK4, activating PDH, and glucose oxidation as the mechanism by which cardiomyocytes remain insulin sensitive.