Eukaryotic Initiation Factor 4E-binding Protein Research Articles

Dietary glycine supplementation activates mTOR 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 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 down-regulated (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 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 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.

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 down-regulated expression of 4e-bp1, a negative regulator of the translation initiation complex. This in turn up-regulates PGC1α protein levels, which 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.

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.

RIP3/MLKL regulates necroptosis via activating 4EBP1-eIF4E pathway.

Necroptosis is a cell death type mediated by receptor interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL). It has been reported that mammalian target of rapamycin plays a regulatory role in necroptosis. Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)-eukaryotic initiation factor 4E (eIF4E) pathway is a key down streamer of mammalian target of rapamycin. However, whether 4EBP1-eIF4E pathway is involved in necroptosis is still unknown. This study aims to investigate the changes of 4EBP1-eIF4E pathway in necroptosis. TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively. After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis.

Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

Eukaryotic initiation factor 5A hypusine (eIF5AHyp) stimulates the translation of proline repeat motifs. Salt inducible kinase 2 (SIK2) containing a proline repeat motif is overexpressed in ovarian cancers, in which it promotes cell proliferation, migration, and invasion. Western blotting and dual luciferase analyses showed that depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA downregulated SIK2 level and decreased luciferase activity in cells transfected with a luciferase-based reporter construct containing consecutive proline residues, whereas the activity of the mutant control reporter construct (replacing P825L, P828H, and P831Q) did not change. According to the MTT assay, GC7, which has a potential antiproliferative effect, reduced the viability of several ovarian cancer cell lines by 20-35% at high concentrations (ES2 > CAOV-3 > OVCAR-3 > TOV-112D) but not at low concentrations. In a pull-down assay, we identified eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP1 (p4E-BP1) phosphorylated at Ser 65 as downstream binding partners of SIK2, and we validated that the level of p4E-BP1(Ser 65) was downregulated by SIK2-targeting siRNA. Conversely, in ES2 cells overexpressing SIK2, the p4E-BP1(Ser65) level was increased but decreased in the presence of GC7 or eIF5A-targeting siRNA. Finally, the migration, clonogenicity, and viability of ES2 ovarian cancer cells were reduced by GC7 treatment as well as by siRNA for eIF5A gene silencing and siRNA for SIK2 and 4E-BP1 gene silencing. Conversely, those activities were increased in cells overexpressing SIK2 or 4E-BP1 and decreased again in the presence of GC7. The depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA attenuated activation of the SIK2-p4EBP1 pathway. In that way, eIF5AHyp depletion reduces the migration, clonogenicity, and viability of ES2 ovarian cancer cells.

Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats

Effects and mechanism of glycine on rat cardiomyocytes pretreated with serum from burned rats

Insights into Overlappings of Fibrosis and Cancer: Exploring the Tumor-related Cardinal Genes in Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is quite similar to that of cancer pathogenesis, and several pathways appear to be involved in both disorders. The mammalian target of the rapamycin (mTOR) pathway harbors several established oncogenes and tumor suppressors. The same signaling molecules and growth factors, such as vascular endothelial growth factor (VEGF), contributing to cancer development and progression play a part in fibroblast proliferation, myofibroblast differentiation, and the production of extracellular matrix in IPF development as well. The expression of candidate genes acting upstream and downstream of mTORC1, as well as Vegf and low-density lipoprotein receptor related protein 1(Lrp1), was assessed using specific primers and quantitative polymerase chain reaction (qPCR) within the lung tissues of bleomycin (BLM)-induced IPF mouse models. Lung fibrosis was evaluated by histological examinations and hydroxyproline colorimetric assay. BLM-exposed mice developed lung injuries characterized by inflammatory manifestations and fibrotic features, along with higher levels of collagen and hydroxyproline. Gene expression analyses indicated a significant elevation of regulatory associated protein of mTOR (Raptor), Ras homolog enriched in brain (Rheb), S6 kinase 1, and Eukaryotic translation initiation factor 4E-binding protein 1 (4Ebp1), as well as a significant reduction of Vegfa, Tuberous sclerosis complex (Tsc2), and Lrp1; no changes were observed in the Tsc1 mRNA level. Our findings support the elevation of S6K1 and 4EBP1 in response to the TSC/RHEB/mTORC1 axis, which profoundly encourages the development and establishment of IPF and cancer. In addition, this study suggests a possible preventive role for VEGF-A and LRP1 in the development of IPF.

Licochalcone A Exerts Anti-Cancer Activity by Inhibiting STAT3 in SKOV3 Human Ovarian Cancer Cells.

Licochalcone A (LicA), a major active component of licorice, has been reported to exhibit various pharmacological actions. The purpose of this study was to investigate the anticancer activity of LicA and detail its molecular mechanisms against ovarian cancer. SKOV3 human ovarian cancer cells were used in this study. Cell viability was measured using a cell counting kit-8 assay. The percentages of apoptotic cells and cell cycle arrest were determined by flow cytometry and Muse flow cytometry. The expression levels of proteins regulating cell apoptosis, cell cycle, and the signal transducer and activator of transcription 3 (STAT3) signaling pathways were examined using Western blotting analysis. The results indicated that LicA treatment inhibited the cell viability of SKOV3 cells and induced G2/M phase arrest. Furthermore, LicA induced an increase in ROS levels, a reduction in mitochondrial membrane potential, and apoptosis accompanied by an increase in cleaved caspases and cytoplasmic cytochrome c. Additionally, LicA caused a dramatic decrease in STAT3 protein levels, but not mRNA levels, in SKOV3 cells. Treatment with LicA also reduced phosphorylation of the mammalian target of rapamycin and eukaryotic translation initiation factor 4E-binding protein in SKOV3 cells. The anti-cancer effects of LicA on SKOV3 cells might be mediated by reduced STAT3 translation and activation.

Mitotic CDK1 and 4E-BP1 II: A single phosphomimetic mutation in 4E-BP1 induces glucose intolerance in mice.

Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation. Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis. Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge. 4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.

The Role of mTOR and Injury in Developing Salispheres

Salispheres are the representative primitive cells of salivary glands grown in vitro in a nonadherent system. In this study, we used the ligation model for salisphere isolation after seven days of obstruction of the main excretory duct of the submandibular gland. The mammalian target of rapamycin (mTOR) is a critical signalling pathway involved in many cellular functions and is suggested to play a role in atrophy. We determined the role of mTOR and injury in the formation and development of salispheres. Morphological assessments and Western blot analysis illustrated how mTOR inhibition by rapamycin impaired the assembly of salispheres and how indirect stimulation of mTOR by lithium chloride (LiCl) assisted in the expansion of the salispheres. The use of rapamycin highlighted the necessity of mTOR for the development of salispheres as it affected the morphology and inhibited the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein (4e-bp1). mTOR activity also appeared to be a crucial regulator for growing salispheres, even from the ligated gland. However, atrophy induced by ductal ligation resulted in a morphological alteration. The phosphorylation of 4e-bp1 and S6 ribosomal protein in cultured salispheres from ligated glands suggests that mTOR was not responsible for the morphological modification, but other unexplored factors were involved. This exploratory study indicates that active mTOR is essential for growing healthy salispheres. In addition, mTOR stimulation by LiCl could effectively play a role in the expansion of salispheres. The impact of atrophy on salispheres suggests a complex mechanism behind the morphological alteration, which requires further investigation.