Phosphorylation Of p70S6K Research Articles

Convergence of signaling pathways in mediating actions of leucine and IGF-1 on mTORC1 in L6 myoblasts.

Leucine and insulin-like growth factor-1 (IGF-1) are important regulators of protein synthesis in skeletal muscle. The mechanistic target of rapamycin complex 1 (mTORC1) is of particular importance in their mechanism of action. In the present study, pathways through which leucine and IGF-1 converge to mediate activation of mTORC1 were examined in L6 myoblasts that were deprived of leucine and serum followed by readdition of either leucine or IGF-1. Compared with leucine- and serum-deprived myoblasts, IGF-1, but not leucine, promoted phosphorylation of protein kinase B (AKT), tuberous sclerosis complex 2 (TSC2), and the autophosphorylation site on mTOR (S2481) and also stimulated mTOR kinase activity in mTOR immunoprecipitated samples. Both leucine and IGF-1 promoted phosphorylation of mTOR on S2448. The association of mTOR with the regulatory-associated protein of mTOR (Raptor) was altered by IGF-1 treatment and trended (P = 0.065) to be altered by leucine treatment. Alterations in the association of mTOR with Raptor were proportional to changes in phosphorylation of the mTOR substrates, eIF4E-binding protein 1 (4E-BP1), and ribosomal protein S6 Kinase-β1 (p70S6K1). Surprisingly, leucine, but not IGF-1, stimulated protein synthesis suggesting a unique role for nutrients in regulating protein synthesis. Overall, the results are consistent with a model whereby IGF-1 stimulates phosphorylation of 4E-BP1 and p70S6K1 in L6 myoblasts through an AKT-TSC2-mTORC1 signaling pathway that also involves changes in the interaction between mTOR and Raptor. In contrast, leucine signaling to mTOR results in alterations in certain mTOR phosphorylation sites and the interaction between mTOR and Raptor and stimulates protein synthesis.

Rapamycin did not prevent the excessive exercise-induced hepatic fat accumulation

AimsThe excessive eccentric exercise led to hepatic fat accumulation, which occurred concomitantly with elevation in the mammalian target of the rapamycin complex 1 (mTORC1) and insulin signaling pathways. Since mTORC1 and insulin can inhibit the autophagy pathway and explain the liver lipid content elevation, the main objective of the present investigation was to verify the responses of genes and proteins related to the autophagy and lipogenesis pathways in the hepatic tissue of mice submitted to the excessive downhill running protocol with and without rapamycin administration, a drug able to inhibit the mTORC1 pathway. Main methodsC57BL/6J mice were divided into four experimental groups: Control (CT; sedentary), Excessive exercise in downhill running (EE), Excessive exercise in downhill running with chronic administration of rapamycin (EE/Rapa), and Endurance exercise (END). At the end of the protocols, the blood and liver were collected for serum analysis, histology, immunohistochemistry, hepatic fat content, reverse transcription-quantitative polymerase chain reaction, and immunoblotting. Key findings1) higher levels of glucose, insulin, HOMA-IR, cortisol, ALT, and cholesterol, but lower levels of T4 for the EE/Rapa group; 2) hepatic fat accumulation for the EE and EE/Rapa groups; 3) upregulation of LC3 immunoexpression and downregulation of autophagic flux index for the EE and EE/Rapa groups; 4) reduction of P70S6K phosphorylation and SQSTM1 and increase of FOXO1A phosphorylation for the EE/Rapa group. SignificanceThe excessive exercise in downhill running with or without rapamycin led to increased liver fat content. Although rapamycin was effective in inhibiting mTORC1, the autophagy flux was not upregulated.

Chip-DSF: A rapid screening strategy for drug protein targets

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip–DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip–DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips.

Loquat Leaf Extract Enhances Muscle Contraction-Induced Activation of Protein Synthesis Signaling in Rat Skeletal Muscle.

Loquat (Eriobotrya japonica (Thunb.) Lindl.) leaves are traditionally used to improve muscle weakness, but their effects on muscle protein synthesis require further research. Therefore, we aimed to investigate whether loquat leaf extract (LLE) enhances muscle contraction-induced activation of muscle protein synthesis signaling. Male Wistar rats (12 weeks old, n = 6/group) were categorized into water treatment (CON) and LLE treatment (LLE) groups. The rats were administered distilled water or LLE (1.5 g/kg/day) once a day by oral gavage for 7 days. On day 7, at 3 h post-LLE administration, the gastrocnemius muscle in the right leg of each rat was stimulated by electrical muscle stimulation (EMS) (100 Hz, 30 V) through five sets of 10 isometric contractions (7 s contraction, 3 s rest) with 3 min interset intervals. The rats were then sacrificed, and the gastrocnemius muscles of both legs were excised at 3 h post-EMS. The phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway molecules (Akt, mTOR, and p70S6K) were determined by Western blotting. Regarding the muscle contraction-induced protein synthesis signaling pathway, Akt phosphorylation at Ser473 was not significantly different between the CON and LLE groups. mTOR phosphorylation at Ser2448 was increased by EMS but did not show a significant difference between the CON and LLE groups. p70S6K phosphorylation at Thr389 was significantly increased in response to EMS, whereas the LLE group showed significantly higher p70S6K phosphorylation at Thr389 than that in the CON group. This suggests that LLE enhances muscle contraction-induced activation of p70S6K phosphorylation in rat skeletal muscles.

Abstract 5495: PHI-501, a potent and novel inhibitor of NRAS mutated acute myeloid leukemia

Abstract Purpose: Development of the first-in-class inhibitor of NRAS mutated acute myeloid leukemia. PHI-501 has potent activity against receptor tyrosine kinases and intracellular kinases related to STAT, AKT, and ERK signaling. Description: Preclinical testing of PHI-501 for efficacy against NRAS mutations both in vitro and in vivo xenografts. Summary: Mutations in the RAS genes (including KRAS, NRAS, and HRAS) are discovered in about 30% of all tumors. KRAS is the most frequently mutated gene in cancers found in pancreatic, colon, and lung, while NRAS mutations around the hot spots at codons 12, 13, and 61 are more common in AML (10.3%). PHI-501 is a novel small molecule inhibitor intended for the treatment of AML in patients expressing the N-RAS activating mutation. PHI-501 presented higher selectivity within a panel of 355 protein kinases. PHI-501 exerts their anti-proliferative effects by inducing apoptosis and G0-G1 arrest and significantly induce apoptotic markers (cleaved PARP and caspase 3) in both Ba/F3-NRAS-G12D and OCI-AML3 cells. The results of Western blot analysis showed that PHI-501 attenuate phosphorylation of p70S6K1, AKT, and p38 in OCI-AML3 After 21 days of the treatment in subcutaneous OCI-AML3 human AML xenograft model, PHI-501 showed significant inhibition of tumor growth (TGI) in a dose-dependent manner. Compared to the vehicle group, the 40 mg/kg group demonstrated a 74.0% lower average tumor volume (P<0.0001) and a 78.5% lower average tumor weight (P=0.0041). No significant body weight changes were observed in any of the groups. In toxicology and pharmacokinetic study, PHI-501 exhibited pharmacokinetic profiles favorable for oral administration in rodent, dog, and monkey and displayed acceptable in vivo and in vitro safety profiles. These results show the significant ability of PHI-501 to inhibit the progression of NRAS activated AML tumor growth, making it a strong candidate for the treatment of AML. Conclusion: The preclinical evaluation of PHI-501, a novel N-RAS inhibitor, showed clear evidence of anticancer activity for AML and improved efficacy in both in vitro and in vivo models. Consequently, PHI-501 is a new potent multi-kinase inhibitor with characteristics that warrant entry into human trials for the treatment of AML in patients expressing the N-RAS activating mutation. Citation Format: Ky-Youb Nam, Jung Hee Park, Kyung Ah Kim, Inje Shin, June H-J Han, Kyu-Tae Kim, Jeong Hyeok Yoon, Hanna Cho, Seung-Hye Choi, Sandip Sengupta, Taebo Sim. PHI-501, a potent and novel inhibitor of NRAS mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5495.

The Effects of Deoxyelephantopin on the Akt/mTOR/P70S6K Signaling Pathway in MCF-7 Breast Carcinoma Cells In Vitro

Objective To determine the effects of deoxyelephantopin on mTOR and its related target molecules (Akt/mTOR/P70S6K) in the ER-positive breast cancer cell line. Materials and Methods Primary in silico simulations were determined, and the effects of deoxyelephantopin on the phosphorylation of the Akt/mTOR/P70S6K molecules were evaluated using AlphaScreen-based assays and western blot analysis, respectively. Results Based on the estimated FEB and K i values, deoxyelephantopin appeared to have a stronger affinity toward P70S6K as compared with Akt and mTOR. Both deoxyelephantopin and control inhibitors were observed to form hydrogen bonds with the same key residue, Leu175 of the P70S6K molecule. Deoxyelephantopin downregulated the p-P70S6K protein expression significantly from 18 µM ( p < 0.05) and onward. Based on the AlphaScreen assay, deoxyelephantopin produced a concentration-dependent inhibition on the phosphorylation of P70S6K with an IC50 value of 7.13 µM. Conclusion Deoxyelephantopin induced cell death in MCF-7 cells possibly via DNA fragmentation, inhibition of the phosphorylation of P70SK6, and downregulation of the relative p-p70S6K protein expression levels.

Orcinol Glucoside Improves Senile Osteoporosis through Attenuating Oxidative Stress and Autophagy of Osteoclast via Activating Nrf2/Keap1 and mTOR Signaling Pathway.

Oxidative stress and autophagy play essential roles in the development of senile osteoporosis which is characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Orcinol glucoside (OG), a phenolic glycoside isolated from Curculigo orchioides Gaertn, possesses antiosteoporotic properties. This study examined the protective effects of OG on bone loss in SAMP6 mice and explored the underlying mechanisms. The osteoporotic SAMP6 mice were treated with OG oral administration. RAW264.7 cells were induced to differentiate into osteoclast by RANKL and H2O2 in vitro and received OG treatment. The results demonstrated that OG attenuated bone loss in SAMP6 mice and inhibited the formation and bone resorption activities of osteoclast and reduced levels of oxidative stress in bone tissue of SAMP6 mice and osteoclast. Furthermore, OG activated Nrf2/Keap1 signaling pathway and enhanced the phosphorylation of mTOR and p70S6K which are consequently suppressing autophagy. Of note, the effect of OG on Nrf2/Keap1 signaling was neutralized by the mTOR inhibitor rapamycin. Meanwhile, the inhibitory effect of OG on autophagy was reversed by the Nrf2 inhibitor ML385.Conclusively, OG attenuated bone loss by inhibiting formation, differentiation, and bone resorption activities of osteoclast. Regulation of Nrf2/Keap1 and mTOR signals is a possible mechanism by which OG suppressed oxidative and autophagy of osteoclasts. Thus, OG prevented senile osteoporosis through attenuating oxidative stress and autophagy of osteoclast via activating Nrf2/Keap1 and mTOR signaling pathway.

Essential Amino Acid Regulation of Protein Turnover to Support Skeletal Muscle Health with Age

Skeletal muscle (SM) is vital for both long term health and quality of life. Recent research suggests an increase in catabolic signals with age triggers pathologic conditions, such as sarcopenia. Although results from in vitro studies model how essential amino acids (EAA) can regulate muscle protein synthesis (MPS), the relevance of these models to muscle protein breakdown (MPB), and the presence of physiological plasma amino acid concentrations (PAA) remain to be established. Therefore, the objective of this study was to determine the effects of EAA with (0.2, 1.0, and 3.0 x PAA) in a young (passages 2‐10) and aging (passages 16‐24) C2C12 murine muscle cell model. We hypothesized that increased levels of EAA will increase MPS in aging cells and suppress MPB via the mTORC1 pathway when compared to young cells. Myoblasts were seeded (1x105) into 6‐well plates and differentiated into myotubes when they reached 80% confluency. Myotubes were serum and AA starved for 24 hours before receiving one of the following treatments: control (CON), 0.2 x PAA, 1.0 x PAA, 3.0 x PAA with or without rapamycin (100 nm; RAP) for 1, 6, and 24 hours. All treatments were performed in triplicate and then each experiment was repeated three times, yielding nine wells per treatment. Proteins related to sensing of AA and protein synthesis and degradation were assessed using Western Blot. Rapamycin inhibited phosphorylation of p70 S6 kinase 1 (p70S6K1) and 4E binding protein 1 (4E‐BP1), downstream targets of mTORC1, in both CON and AA‐treated young and aged cells. Phosphorylation of 4EBP1 and p70S6K1 was significantly increased (p<0.05) in both young and aged cells following all PAA treatments. However, there was no significance after 24 hours of AA supplementation in aged cells. Particularly, after 6 hours of treatment all doses significantly increased (p<0.0001) phosphorylation of p70S6K1 in both young and aged cells. 0.2 x PAA and 3.0 x PAA elicited a significantly greater response in 4EBP1 phosphorylation in young cells compared to the CON after 6 hours of treatment, whereas a dose of 3.0 x PAA induced a similar response in aged cells. In conclusion, in a C2C12 myotube model, increasing EAA concentrations downregulates the expression of translation initiation factors in young and aging cells. Specifically, it takes a 3.0 x PAA to elicit a response in aging cells, whereas young cells respond to lower physiological concentrations of 0.2 x PAA and 1.0 x PAA. Therefore, it can be proposed increasing EAA may be effective for adjusting the rate of MPS and MPB via the mTORC1 pathway in both young and aging muscle cells.

ALDH2 Deficiency Enhances Mechanical Overload‐Induced Protein Synthesis but Not Muscle Mass

Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme of redox homeostasis and ethanol metabolism. Our recent work has indicated an important role for ALDH2 in regulating muscle homeostasis as well. However, the underlying mechanism and adaptability of ALDH2 in skeletal muscle remain highly elusive. We herein examined whether ALDH2 deficiency modifies mechanical overload‐induced muscle hypertrophy and related cellular process. Ten‐week‐old male ALDH2 knockout (background strain: C57BL/6J) and wild‐type littermates were involved in this work. Unilateral synergist ablation (SA; the surgical removal of gastrocnemius and soleus muscles) and sham surgery were performed as an experimental hypertrophy model. Seven days after surgery, plantaris muscles were collected. ALDH2 deficiency augmented SA‐induced activation of mTORC1 (a key regulator of muscle anabolism), based on the increase in phosphorylation of p70S6K at Thr389. Concomitantly, higher amounts of puromycin‐labeled peptides (a marker of protein synthesis rate) were observed in ALDH2 deficit muscle. These observations suggest that ALDH2 deficiency promotes SA‐induced muscle hypertrophy. Unexpectedly, there was no statistical difference in SA‐induced muscle hypertrophy between genotypes. To explore potential mechanisms underlying mismatch between protein synthesis signal and muscle size, we conducted RNA‐seq experiment. However, we couldn’t detect any notable gene potentially affecting muscle mass between genotypes. In conclusion, ALDH2 deficiency enhances mechanical overload‐induced mTORC1 activity and protein synthesis but not muscle mass. Pathways other than protein synthesis and/or post‐transcriptional events may dominantly affect muscle mass in ALDH2 deficit mice.

Rapamycin Suppresses Penile NADPH Oxidase Activity to Preserve Erectile Function in Mice Fed a Western Style High‐Fat, High‐Sucrose Diet

IntroductionThe mechanistic target of rapamycin (mTOR) is a nutrient‐sensitive cellular signaling kinase that has been shown to be activated by high‐fat and/or high‐sugar feeding in highly metabolic tissues such as adipose tissue, liver, and skeletal muscle. mTOR activation has been implicated in the excess production of reactive oxygen species (ROS) resulting from overnutrition, which has been associated with activation of NADPH oxidase (Nox) as a source of excess ROS production. A Western style high‐fat, high‐sucrose diet has recently been used to induce erectile dysfunction (ED) in rodents, in which elevated Nox has been implicated in ED pathogenesis. The objective of this study was to determine if mTOR is an upstream mediator of Nox in the penis in response to the Western diet (WD) and to determine if this pathway is relevant in WD‐induced ED.MethodsYoung male C57Bl/6 mice (n = 90) were fed a control diet (CD) or WD ad libitum for 12 weeks. For the final four weeks of the dietary intervention, mice were intraperitoneally injected with either vehicle (Veh) or the mTOR inhibitor rapamycin (Rap; 2 mg/kg) three days/week. Following the intervention, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation. In separate mice following the same intervention, in vivo ROS production was measured in the penis utilizing a microdialysis approach. Microdialysis probes were inserted into the penis of anesthetized mice and perfused with saline containing 100 μM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase, and fluorescence was measured from three dialysate replicates. 300 μM apocynin, a Nox inhibitor, was then added to the perfusate, and additional dialysate samples were analyzed. Nox‐mediated ROS were determined by calculation of the ROS that was inhibited by apocynin. Western blots were performed on corpus cavernosum (CC) tissue for Nox subunits, phosphorylation of the mTOR active site, and phosphorylation of p70S6K, a downstream mediator of mTOR Complex 1 (mTORC1) signaling. Significant differences between groups were determined by two‐way ANOVA with Tukey’s multiple comparisons post‐hoc analysis.ResultsErectile function was significantly impaired in mice fed the WD in the vehicle condition, while Rap restored erectile function in the WD condition. P‐mTOR/mTOR and P‐p70S6K/p70S6K were increased in CC from WD‐Veh relative to CD‐Veh mice, while both of these measures were markedly suppressed by Rap treatment regardless of diet. Penile production of the ROS hydrogen peroxide and superoxide were elevated in vehicle treated WD‐fed mice, which was normalized in WD‐fed mice with Rap treatment. Similarly, Nox‐mediated penile ROS was elevated in vehicle treated WD‐fed mice, which was normalized in WD‐fed mice with Rap treatment. The Nox subunits Nox2, p47, and p22 were significantly elevated in CC from WD‐Veh mice, while Rap significantly blunted these expressions. The p67 and Nox4 subunits were not different amongst any groups.ConclusionsThe mTORC1/p70S6K axis appears to be an upstream mediator of Nox2 in the corpus cavernosum in response to chronic consumption of a high‐fat, high‐sucrose, Western pattern diet, which has a deleterious effect on erectile function.

Effects of a novel AMP‐mimicking prodrug on AMPK and mTORC1 activity in C2C12 cells

The beneficial effects of exercise on skeletal muscle health and function are well‐established. The intracellular signaling that mediates exercise’s effects on muscle are undoubtedly complex and are not entirely understood. Nonetheless, increasing adenosine monophosphate (AMP) levels during muscle contraction signals a declining energy charge in the cell and this certainly drives many of the beneficial adaptations of exercise. AMP‐activated protein kinase (AMPK) is one of AMP’s best‐defined targets and has been described as the cell’s master energy regulator. Because it is activated by exercise, AMPK has gained recent attention as an attractive pharmacological target in harnessing at least some of the benefits of exercise for those who cannot exercise due to old age or other disorders. The prodrug AICAR effectively activates AMPK as it is taken up into the cell and phosphorylated to form ZMP, an AMP mimetic. However, AICAR’s therapeutic potential is severely limited by its poor pharmacokinetics. Here, we describe a novel approach to ZMP delivery in a novel prodrug (Prodrug‐39; P39). We found that similar to AICAR, P39 concentrations above ~400 μM effectively achieved significant AMPK activation and glycogen depletion. Surprisingly, given AMPK’s well‐documented inhibition of the mechanistic target of rapamycin (mTORC1), P39 was also effective at stimulating p70S6k and rpS6 phosphorylation concentrations as low as 10 uM and early in the timecourse after administration of P39 at higher concentrations. Given this evidence, we conclude that Prodrug‐39 is indeed an effective AMPK activator and may also have some utility in mTOR activation at lower concentrations.

Lack of Tgfbr1 and Acvr1b synergistically stimulates myofibre hypertrophy and accelerates muscle regeneration.

In skeletal muscle, transforming growth factor-β (TGF-β) family growth factors, TGF-β1 and myostatin, are involved in atrophy and muscle wasting disorders. Simultaneous interference with their signalling pathways may improve muscle function; however, little is known about their individual and combined receptor signalling. Here, we show that inhibition of TGF-β signalling by simultaneous muscle-specific knockout of TGF-β type I receptors Tgfbr1 and Acvr1b in mice, induces substantial hypertrophy, while such effect does not occur by single receptor knockout. Hypertrophy is induced by increased phosphorylation of Akt and p70S6K and reduced E3 ligases expression, while myonuclear number remains unaltered. Combined knockout of both TGF-β type I receptors increases the number of satellite cells, macrophages and improves regeneration post cardiotoxin-induced injury by stimulating myogenic differentiation. Extra cellular matrix gene expression is exclusively elevated in muscle with combined receptor knockout. Tgfbr1 and Acvr1b are synergistically involved in regulation of myofibre size, regeneration, and collagen deposition.

Tripartite motif 27 promotes cardiac hypertrophy via PTEN/Akt/mTOR signal pathways

ABSTRACT Tripartite motif-containing 27 (Trim27) is highly expressed in tumor cells and regulates natural immunity and apoptosis. However, the effects of Trim27 in cardiac hypertrophy are not fully elucidated. In this study, we explored the potential role of Trim27 in pressure overload-induced cardiac hypertrophy and the underlying mechanism. The results indicated that compared to sham operation (Sham) group, transverse aortic constriction (TAC) group showed significantly up-regulated Trim27 protein expression (P < 0.05). The neonatal rat cardiomyocytes (NRCMs) were isolated and stimulated with PBS, angiotensin (AngII) and phenylephrine (PE). NRCMs were collected to detect the protein expression of Trim27. The results were consistent with the results in vivo. Compared to PBS treatment, the expression of Trim27 protein in NRCMs was significantly increased after PE or AngII stimulation (P < 0.05, respectively). Knockout of Trim27 reduced the size of cardiomyocytes and the protein expression of ANP, BNP, and β-MHC, improved cardiac function, and decreased myocardial hypertrophy (P < 0.05). Trim27 may be involved in regulating the development of cardiac hypertrophy. Further results showed that Trim27 enhanced the protein expression of phosphorylation of Akt, GSK3β, mTOR, and P70s6k by interacting with PTEN (phosphatase tensin homolog). These findings revealed that Trim27 can promote cardiac hypertrophy by activating PTEN/Akt/GSK3β/mTOR signaling pathways.

Mild Endurance Exercise during Fasting Increases Gastrocnemius Muscle and Prefrontal Cortex Thyroid Hormone Levels through Differential BHB and BCAA-Mediated BDNF-mTOR Signaling in Rats.

Mild endurance exercise has been shown to compensate for declined muscle quality and may positively affect the brain under conditions of energy restriction. Whether this involves brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) activation in relation to central and peripheral tissue levels of associated factors such as beta hydroxy butyrate (BHB), branched-chain amino acids (BCAA) and thyroid hormone (T3) has not been studied. Thus, a subset of male Wistar rats housed at thermoneutrality that were fed or fasted was submitted to 30-min-mild treadmill exercise bouts (five in total, twice daily, 15 m/min, 0° inclination) over a period of 66 h. Prefrontal cortex and gastrocnemius muscle BHB, BCAA, and thyroid hormone were measured by LC-MS/MS analysis and were related to BDNF and mammalian target of rapamycin (mTOR) signaling. In gastrocnemius muscle, mild endurance exercise during fasting maintained the fasting-induced elevated BHB levels and BDNF-CREB activity and unlocked the downstream Akt-mTORC1 pathway associated with increased tissue BCAA. Consequently, deiodinase 3 mRNA levels decreased whereas increased phosphorylation of the mTORC2 target FOXO1 was associated with increased deiodinase 2 mRNA levels, accounting for the increased T3 tissue levels. These events were related to increased expression of CREB and T3 target genes beneficial for muscle quality previously observed in this condition. In rat L6 myoblasts, BHB directly induced BDNF transcription and maturation. Mild endurance exercise during fasting did not increase prefrontal cortex BHB levels nor was BDNF activated, whereas increased leucine levels were associated with Akt-independent increased phosphorylation of the mTORC1 target P70S6K. The associated increased T3 levels modulated the expression of known T3-target genes involved in brain tissue maintenance. Our observation that mild endurance exercise modulates BDNF, mTOR and T3 during fasting provides molecular clues to explain the observed beneficial effects of mild endurance exercise in settings of energy restriction.

Circadian rhythms modulate the effect of eccentric exercise on rat soleus muscles.

We investigated whether time-of-day dependent changes in the rat soleus (SOL) muscle size, after eccentric exercises, operate via the mechanistic target of rapamycin (mTOR) signaling pathway. For our first experiment, we assigned 9-week-old male Wistar rats randomly into four groups: light phase (zeitgeber time; ZT6) non-trained control, dark phase (ZT18) non-trained control, light phase-trained, and dark phase-trained. Trained animals performed 90 min of downhill running once every 3 d for 8 weeks. The second experiment involved dividing 9-week-old male Wistar rats to control and exercise groups. The latter were subjected to 15 min of downhill running at ZT6 and ZT18. The absolute (+12.8%) and relative (+9.4%) SOL muscle weights were higher in the light phase-trained group. p70S6K phosphorylation ratio was 42.6% higher in the SOL muscle of rats that had exercised only in light (non-trained ZT6). Collectively, the degree of muscle hypertrophy in SOL is time-of-day dependent, perhaps via the mTOR/p70S6K signaling.

BDK knockout skeletal muscle satellite cells exhibit enhanced protein translation initiation signal in response to BCAA in vitro

We examined the effects of branched-chain amino acids (BCAAs) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.

Comprehensive analysis of transcriptomics and metabolomics to illustrate the underlying mechanism of helenalin against hepatic fibrosis

This study aimed to investigate the protective mechanisms of helenalin on hepatic fibrosis. In brief, rats were intragastrically administrated with 50% CCl4 for 9 weeks to induce liver fibrosis, followed by treatment with various agents for 6 weeks. The effects of helenalin on hepatic injury were assessed by pathological examinations. The potential targets were predicted by the “Drug-Disease” bioinformatic analysis and then verified by multiple experiments. Moreover, the underlying mechanism was investigated by transcriptomics and metabolomics as a whole. The results showed that helenalin significantly alleviated hepatocyte necrosis and hepatic injury, as proved by the pathological examinations. Also, helenalin markedly attenuated hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. Besides, helenalin could significantly reduce collagen accumulation, as evidenced by the decreased contents of collagen, hyaluronic acid and laminin. Moreover, helenalin significantly down-regulated the phosphorylation of PI3K, Akt, FAK, mTOR and P70S6K, and PTEN protein expression, suggesting that helenalin inhibited the PI3K/Akt signaling cascade. Meanwhile, helenalin inhibited the NF-κB signaling pathway by reducing the phosphorylation of IκBα, NF-κB p65 and IKKα/β, alleviating inflammation response. Interestingly, the analysis of transcriptomics and metabolomics indicated that helenalin inhibited the glycerophospholipid metabolism pathway by down-regulating the target genes (CHKA, ETNPPL, LYPLA1, PCYT2, PLD4 and PNPLA6), ultimately ameliorating hepatocyte damage. In conclusion, helenalin ameliorates hepatic fibrosis by regulating the PI3K/Akt and NF-κB signaling pathways and the glycerophospholipid metabolism pathway.

Novel Repositioning Therapy for Drug-Resistant Glioblastoma: In Vivo Validation Study of Clindamycin Treatment Targeting the mTOR Pathway and Combination Therapy with Temozolomide.

Simple SummaryGiven the significant costs and lengthy timelines of drug development and clinical trials, drug repositioning is a promising alternative to find effective treatments for brain tumors quickly and inexpensively. In the present study, using a simple drug screen of macrolides, we found that clindamycin (CLD) had cytotoxic effects on glioblastoma (GBM) cells. Further studies showed the inhibition of the mammalian target of rapamycin (mTOR) pathway as the key mechanism of action. Interestingly, we found that co-treatment with temozolomide (TMZ), the alkylating agent considered as standard therapy in GBM, enhanced these effects and proposed the inhibition of O6-methylguanine-DNA methyltransferase (MGMT) protein by CLD as a potential mechanism for this combination effect.Multimodal therapy including surgery, radiation treatment, and temozolomide (TMZ) is performed on glioblastoma (GBM). However, the prognosis is still poor and there is an urgent need to develop effective treatments to improve survival. Molecular biological analysis was conducted to examine the signal activation patterns in GBM specimens and remains an open problem. Advanced macrolides, such as azithromycin, reduce the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream mammalian target of rapamycin (mTOR) effector, and suppress the proliferation of T-cells. We focused on its unique profile and screened for the antitumor activity of approved macrolide antibiotics. Clindamycin (CLD) reduced the viability of GBM cells in vitro. We assessed the effects of the candidate macrolide on the mTOR pathway through Western blotting. CLD attenuated p70S6K phosphorylation in a dose-dependent manner. These effects on GBM cells were enhanced by co-treatment with TMZ. Furthermore, CLD inhibited the expression of the O6-methylguanine-DNA methyltransferase (MGMT) protein in cultured cells. In the mouse xenograft model, CLD and TMZ co-administration significantly suppressed the tumor growth and markedly decreased the number of Ki-67 (clone MIB-1)-positive cells within the tumor. These results suggest that CLD suppressed GBM cell growth by inhibiting mTOR signaling. Moreover, CLD and TMZ showed promising synergistic antitumor activity.

Dimethyl Fumarate Combined With Vemurafenib Enhances Anti-Melanoma Efficacy via Inhibiting the Hippo/YAP, NRF2-ARE, and AKT/mTOR/ERK Pathways in A375 Melanoma Cells.

Melanoma is a deadly form of skin cancer with high rates of resistance to traditional chemotherapy and radiotherapy. BRAF inhibitors (BRAFi) can achieve initial efficacy when used to treat melanoma patients, but drug resistance and relapse are common, emphasizing the need for new therapeutic strategies. Herein, we reported that combination of dimethyl fumarate (DMF) and vemurafenib (Vem) inhibited melanoma cell proliferation more significantly and induced more cell death than single agent did both in vitro and in vivo. DMF/Vem treatment induced cell death through inhibiting the expression and transcriptional activity of NRF2 thereby resulting in more reactive oxygen species (ROS) and via inhibiting the expression of YAP, a key downstream effector of Hippo pathway. DMF/Vem treatment also reduced phosphorylation of AKT, 4EBP1, P70S6K and ERK in AKT/mTOR/ERK signaling pathways. RNA-seq analysis revealed that DMF/Vem treatment specifically suppressed 4561 genes which belong to dozens of cell signaling pathways. These results indicated that DMF/Vem treatment manifested an enhanced antitumor efficacy through inhibiting multiple cell signaling pathways, and thus would be a novel promising therapeutic approach targeted for melanoma.

Fisetin Attenuates Diabetic Nephropathy-Induced Podocyte Injury by Inhibiting NLRP3 Inflammasome.

Diabetic nephropathy (DN) is one of the primary complications of diabetes. Fisetin is a flavonoid polyphenol that is present in several vegetables and fruits. The present study investigated the mechanisms of fisetin in DN-induced podocyte injury both in vitro and in vivo. The results revealed that fisetin ameliorated high glucose (HG)-induced podocyte injury and streptozotocin (STZ)-induced DN in mice. CDKN1B mRNA expression in the glomeruli of patients with DN decreased based on the Nephroseq dataset, and fisetin reversed CDKN1B expression at mRNA and protein levels in a dose-dependent manner in podocytes and mice kidney tissues. Furthermore, fisetin suppressed the phosphorylation of P70S6K, a downstream target of CDKN1B, activated autophagosome formation, and inhibited Nod-like receptor protein 3 (NLRP3) inflammasomes. Interfering CDKN1B reduced the protective effects of fisetin against high glucose-induced podocyte injury. Molecular docking results revealed a potential interaction between fisetin and CDKN1B. In summary, the present study revealed that fisetin alleviated high glucose-induced podocyte injury and STZ-induced DN in mice by restoring autophagy-mediated CDKN1B/P70S6K pathway and inhibiting NLRP3 inflammasome.