S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

Radiation is a mainstay of lung cancer treatment; however, resistance frequently develops. Identifying novel therapeutic targets to increase radiation sensitivity is crucial. S6K1 is a serine/threonine kinase known to regulate protein translation which is associated with radioresistance, but the mechanisms involved are unknown. We proposed to determine whether S6K1 promotes radioresistance by regulating DNA repair in lung cancer. Colony formation, protein expression and proliferation were assessed. S6K1 was modulated pharmacologically by either PF-4708671 or genetically by Crispr-Cas9. Higher radioresistance levels in lung cancer cells were associated with lower phosphoactivation of MRN complex members, a key activator of radiation-induced DNA repair signaling. We also found lower levels of p-ATM, a target of the MRN complex, in more radioresistant cells, which was associated with a lower expression of γ-H2AX cafter radiation. Further, genetic and pharmacological S6K1 targeting sensitized lung cancer cells to low doses of radiation (p ≤ 0.01). Additionally, S6K1-/- deletion increased the phosphoactivation of MRN complex members, indicating that S6K1 itself can shut down DNA damage regulated by MRN signaling. This is the first report showing that S6K1 inhibition radiosensitizes lung cancer cells by decreasing MRN complex-regulated DNA repair signaling. Future studies should evaluate the role of S6K1 as a target to overcome radioresistance.

Lactate-upregulated ARG2 expression induces cellular senescence in fibroblast-like synoviocytes of osteoarthritis via activating the mTOR/S6K1 signaling pathway.

Cellular senescence was implicated in the pathogenesis of age-related diseases such as osteoarthritis (OA). Increasing evidence suggests that alterations in the OA joint microenvironment play a crucial role in the pathogenesis of OA. This study aims to establish a clear link between the impact of accumulated lactate on the senescence of fibroblast-like synoviocytes (FLS) within the OA microenvironment. OA models and models with intra-articular injection of lactate were established in rat models, histological analyses were performed. Human OA-FLS treated with lactate was analyzed by mRNA sequencing, senescence related experiments and underlying signaling pathway activation were comprehensively evaluated. This study confirmed that OA models and lactate-injection models exhibited higher synovitis scores. Enrichment analyses indicated dysregulated cell cycle and cellular senescence pathways in OA-FLS treated with lactate. Lactate significantly up-regulated arginase 2 (ARG2) expression and promoted OA-FLS senescence, including G1/S arrest, increased reactive oxygen species and ß-galactosidase production, high expression of senescence-associated secretory phenotype factors, which could be attenuated by siRNA-Arg2. The ARG2-mTOR/S6K1 axis was identified as a potential signaling for lactate-induced OA-FLS senescence, and activated mTOR/S6K1 signaling could be reduced by siRNA-Arg2, rapamycin (mTOR inhibitor), and LY294002 (PI3K inhibitor). Our study provides novel targets and insights for OA therapies.

Coordinated Targeting of S6K1/2 and AXL Disrupts Pyrimidine Biosynthesis in PTEN-Deficient Glioblastoma.

Intrinsic resistance to targeted therapeutics in PTEN-deficient glioblastoma (GBM) is mediated by redundant signaling networks that sustain critical metabolic functions. Here, we demonstrate that coordinated inhibition of the ribosomal protein S6 kinase 1 (S6K1) and the receptor tyrosine kinase AXL using LY-2584702 and BMS-777607 can overcome network redundancy to reduce GBM tumor growth. This combination of S6K1 and AXL inhibition suppressed glucose flux to pyrimidine biosynthesis. Genetic inactivation studies to map the signaling network indicated that both S6K1 and S6K2 transmit growth signals in PTEN-deficient GBM. Kinome-wide ATP binding analysis in inhibitor-treated cells revealed that LY-2584702 directly inhibited S6K1, and substrate phosphorylation studies showed that BMS-777607 inactivation of upstream AXL collaborated to reduce S6K2-mediated signal transduction. Thus, combination targeting of S6K1 and AXL provides a kinase-directed therapeutic approach that circumvents signal transduction redundancy to interrupt metabolic function and reduce growth of PTEN-deficient GBM. SIGNIFICANCE: Therapy for glioblastoma would be advanced by incorporating molecularly targeted kinase-directed agents, similar to standard of care strategies in other tumor types. Here, we identify a kinase targeting approach to inhibit the metabolism and growth of glioblastoma.

Effect of Oral Skim Milk Administration on Skeletal Muscle Protein Synthesis after Total Gastrectomy in Rat.

Leucine is a branched-chain amino acid that is present in protein, and it is an essential factor in activating the mechanistic target of the rapamycin complex 1 signaling pathway and increasing muscle protein synthesis. However, the loss of digestive function after total gastrectomy leads to impaired protein absorption, potentially failing to stimulate muscle protein synthesis. Therefore, this study aimed to investigate whether muscle protein synthesis is enhanced by oral skim milk administration after total gastrectomy. Male Sprague Dawley rats were divided into total gastrectomy (TG) and sham surgery (S) groups. After five weeks postoperatively, we orally administered skim milk to achieve 3.1 g protein/kg body weight and collected blood and gastrocnemius muscle. The gastrocnemius muscle weight was significantly lower in the TG group than in the S group (p < 0.05). The increase in plasma leucine concentration was significantly lower in the TG group than in the S group (p < 0.05). The skeletal muscle protein synthesis and the phosphorylation of p70S6K and 4E-BP1 showed a similar increase in both groups. Even after TG, muscle protein synthesis was stimulated by consuming skim milk, accompanied by a sufficient rise in plasma leucine concentration.

Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation.

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

mTORC1 activation in presumed classical monocytes: observed correlation with human size variation and neuropsychiatric disease.

BACKGROUND: Gain of function disturbances in nutrient sensing are likely the largest component in human age-related disease. Mammalian target of rapamycin complex 1 (mTORC1) activity affects health span and longevity. The drugs ketamine and rapamycin are effective against chronic pain and depression, and both affect mTORC1 activity. Our objective was to measure phosphorylated p70S6K, a marker for mTORC1 activity, in individuals with psychiatric disease to determine whether phosphorylated p70S6K could predict medication response. METHODS: Twenty-seven females provided blood samples in which p70S6K and phosphorylated p70S6K were analyzed. Chart review gathered biometric measurements, clinical phenotypes, and medication response. Questionnaires assessed anxiety, depression, autism traits, and mitochondrial dysfunction, to determine neuropsychiatric disease profiles. Univariate and multivariate statistical analyses were used to identify predictors of medication response. RESULTS: mTORC1 activity correlated highly with both classical biometrics (height, macrocephaly, pupil distance) and specific neuropsychiatric disease profiles (anxiety and autism). Across all cases, phosphorylated p70S6K was the best predictor for ketamine response, and also the best predictor for rapamycin response in a single instance. CONCLUSIONS: The data illustrate the importance of mTORC1 activity in both observable body structure and medication response. This report suggests that a simple assay may allow cost-effective prediction of medication response.

Insulin and IGF-1 extend the lifespan of Caenorhabditis elegans by inhibiting insulin/insulin-like signaling and mTOR signaling pathways: C. elegans - Focused cancer research.

The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.

A strategy for improving silk yield and organ size in silk-producing insects.

Insect silks possess excellent biodegradability, biocompatibility and mechanical properties, and have numerous applications in biomedicine and tissue engineering. However, the application of silk fiber is hindered by its limited supply, especially from non-domesticated insects. In the present study, the silk yield and organ size of Bombyx mori were significantly improved through genetic manipulation of the target of rapamycin complex 1 (TORC1) pathway components. Silk protein synthesis and silk gland size were decreased following rapamycin treatment, inhibiting the TORC1 signaling pathway both in vivo and ex vivo. The overexpression of posterior silk gland-specific Rheb and BmSLC7A5 improved silk protein synthesis and silk gland size by activating the TORC1 signaling pathway. Silk yield in BmSLC7A5-overexpression silkworms was significantly increased by approximately 25%. We have demonstrated that the TORC1 signaling pathway is involved in the transcription and translation of silk genes and transcriptional activators via phosphorylation of p70 S6 kinase 1 and 4E-binding protein 1. Our findings present a strategy for increasing silk yield and organ size in silk-producing insects.

Targeting S6K/NFκB/SQSTM1/Polθ signaling to suppress radiation resistance in prostate cancer.

In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.

YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway.

Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.

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 1 wk 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.

Soybean meal is the major source of dietary protein for growing pigs in many regions of the world, including North America, South America, and Asia. However, this conventional feedstuff is recognized to be severely deficient in glycine (the most abundant amino acid in the bodies of animals, including pigs). Compared to pigs with normal birth weights (NBW), pigs with intrauterine growth restriction (IUGR) have a lower ability to synthesize glycine and reduced growth performance between weaning and market weights. Results of recent studies with cultured muscle cells have revealed that glycine stimulates the mechanistic target of rapamycin (mTOR) cell signaling pathway (the master activator of initiation of protein synthesis in tissues) to promote protein synthesis and cell growth. There is also evidence that the mTOR pathway is impaired in the skeletal muscle of young IUGR pigs. Thus, dietary glycine supplementation may play an important role in maintaining an active mTOR cell signaling pathway for the growth of tissues, particularly skeletal muscle. Results of this study indicated that market-weight IUGR pigs had lower abundances of both total and phosphorylated mTOR, as well as its downstream target proteins in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum, when compared with NBW pigs. In contrast, neither IUGR nor glycine supplementation affected the mTOR cell signaling pathway in the liver of pigs. Taken together, these novel findings indicate that IUGR pigs have insufficient cell signaling via the mTOR cell pathway in a tissue-specific manner during their growing-finishing phases of development and that this negative impact of IUGR can be mitigated by supplementing corn- and soybean meal-based diets with 1% glycine.

High salt diet induces cognitive impairment and is linked to the activation of IGF1R/mTOR/p70S6K signaling.

A high-salt diet (HSD) has been associated with various health issues, including hypertension and cardiovascular diseases. However, recent studies have revealed a potential link between high salt intake and cognitive impairment. This study aims to investigate the effects of high salt intake on autophagy, tau protein hyperphosphorylation, and synaptic function and their potential associations with cognitive impairment. To explore these mechanisms, 8-month-old male C57BL/6 mice were fed either a normal diet (0.4% NaCl) or an HSD (8% NaCl) for 3 months, and Neuro-2a cells were incubated with normal medium or NaCl medium (80 mM). Behavioral tests revealed learning and memory deficits in mice fed the HSD. We further discovered that the HSD decreased autophagy, as indicated by diminished levels of the autophagy-associated proteins Beclin-1 and LC3, along with an elevated p62 protein level. HSD feeding significantly decreased insulin-like growth factor-1 receptor (IGF1R) expression in the brain of C57BL/6 mice and activated mechanistic target of rapamycin (mTOR) signaling. In addition, the HSD reduced synaptophysin and postsynaptic density protein 95 (PSD95) expression in the hippocampus and caused synaptic loss in mice. We also found amyloid ß accumulation and hyperphosphorylation of tau protein at different loci both in vivo and in vitro. Overall, this study highlights the clinical significance of understanding the impact of an HSD on cognitive function. By targeting the IGF1R/mTOR/p70S6K pathway or promoting autophagy, it may be possible to mitigate the negative effects of high salt intake on cognitive function.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Oncostatin M suppresses bone morphogenetic protein-4-induced osteoprotegerin synthesis in MC3T3-E1 osteoblast-like cells: p70 S6 kinase attenuation.

Evidence is accumulating that osteal macrophages, in addition to bone-resorbing osteoclasts and bone-forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in MC3T3-E1 osteoblast-like cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP-4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells. Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP-4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblast-like cells.

mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

Quercetin induces ferroptosis by inactivating mTOR/S6KP70 pathway in oral squamous cell carcinoma.

Although recent studies increasingly suggest the potential anti-cancer effect of quercetin, the exact underlying mechanism remains poorly demonstrated in oral squamous cell carcinoma (oSCC). Therefore, our research explored the impacts of quercetin on the ferroptosis and mTOR/S6KP70 axis in oSCC cell lines. After treating oSCC cells with quercetin or indicated compounds and transfection with SLC7A11- or S6KP70-overexpressing plasmid, cell viability was detected by CCK-8 assay. The level of ferroptosis in oSCC cells was assessed by measuring ROS and GSH levels. The activation of mTOR/S6KP70 axis was assessed by Western blotting. Quercetin promoted ferroptosis in an mTOR/S6KP70-dependent manner to inhibit tumor growth in oSCC cells. Mechanistically, we revealed that quercetin induced lipid peroxidation and reduced GSH levels by repressing SLC7A11 expression in oSCC cells. Specifically, the effects of quercetin on ferroptosis and mTOR and S6KP70 phosphorylation were partially blocked by both mTOR agonist and S6KP70 overexpression. Moreover, mTOR inhibitor promoted ferroptosis in quercetin-treated oSCC cells. Our findings showed that ferroptosis may be a new anti-tumor mechanism of quercetin. Additionally, we identified that quercetin can target mTOR/S6KP70 cascade to inhibit the growth of oSCC cells.

ß-Hydroxy-ß-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes.

ß-Hydroxy-ß-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.

Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells.

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

Ginsenoside Rg1 Induces Autophagy in Colorectal Cancer through Inhibition of the Akt/mTOR/p70S6K Pathway.

This study aimed to elucidate the anti-colon cancer mechanism of ginsenoside Rg1 in vitro and in vivo. Cell viability rate was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay. The inhibitory effect of ginsenoside Rg1 against CT26 cell proliferation gradually increased with increasing concentration. The in vivo experiments also demonstrated an antitumor effect. The monodansylcadaverine (MDC), transmission electron microscopy (TEM), and expression of autophagy marker proteins confirmed that ginsenoside Rg1 induced autophagy in vitro. Ginsenoside Rg1 induced autophagy death of CT26 cells, but this effect could be diminished by autophagy inhibitor (3-methyladenine, 3-MA). Additionally, in a xenograft model, immunohistochemical analysis of tumor tissues showed that the LC3 and Beclin-1 proteins were highly expressed in the tumors from the ginsenoside Rg1-treated nude mice, confirming that ginsenoside Rg1 also induced autophagy in vivo. Furthermoer, both in vivo and in vitro, the protein expressions of p-Akt, p-mTOR, and p-p70S6K were inhibited by ginsenoside Rg1, which was verified by Akt inhibitors. These results indicated that the mechanism of ginsenoside Rg1 against colon cancer was associated with autophagy through inhibition of the Akt/mTOR/p70S6K signaling pathway.

Female Protection Against Diabetic Kidney Disease Is Regulated by Kidney-Specific AMPK Activity.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.