P70 Ribosomal Protein S6 Kinase Research Articles

2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-β (TGF-β)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

Anabolic Signaling After Withdrawl of AMPK-Activating Drugs in C2C12 Cells

Adenosine monophosphate-activated protein kinase (AMPK) is a master metabolic regulator that is activated under low-energy conditions, which generally promotes catabolic processes, including an increase of ATP synthesis and inhibiting anabolic processes that consume ATP. Indeed, acute AMPK activation potently inhibits anabolic pathways, including Akt/mTORC1 signaling. However, recent research reported that withdrawal of the AMPK-activating drug AICAR after a short treatment period results in enhanced anabolic signaling through Akt/mTORC1. This work aims to extend those findings using a more specific AMPK activator since AICAR is well-known to have numerous AMPK-independent effects. To do so, we treated differentiated C2C12 cells with either 2 mM AICAR, 5uM MK8722 (a pan-AMPK activator), or vehicle control (DMSO) for 60 minutes. After 60 minutes of incubation with the drugs, the treatment media was removed, the cells were rinsed and lysed immediately or incubated with media without drug treatment for 30, 60, 120, or 180 minutes, and then lysed. Phosphorylation of ACC (as an indicator of AMPK activity) and anabolic markers Akt, p70 ribosomal protein S6 kinase (S6k), and eIF4E-binding protein (4EBP1) were measured by western blotting. ACC phosphorylation (reflective of AMPK activity) remained elevated through 180 minutes after drug withdrawal for both AICAR and MK8722. Both AKT and S6k phosphorylation remained suppressed for at least 60 minutes after withdrawal of both AICAR and MK8722 and were not elevated at any time point up to 3 hours after withdrawal of the drugs. In contrast to previously reported data, we conclude that anabolic signaling remains inhibited in skeletal muscle myotubes for up to 3 hours after 1 hour of pharmacological activation of AMPK. This work was funded by a BYU Gerontology Grant. 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.

The involvement of Akt, mTOR, and S6K in the in vivo effect of IGF-1 on the regulation of rat cardiac Na+/K+-ATPase.

We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50μg/kg) for 24h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.

The Therapeutic Efficacy and Mechanism of Action of Gnetin C, a Natural Compound from the Melinjo Plant, in a Preclinical Mouse Model of Advanced Prostate Cancer.

The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26MTA1; Ptenf/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future.

Effects of lactic acid bacteria on protein metabolism of Sunit lamb

To investigate the effect of adding lactic acid bacteria (LAB) to Sunit lambs’ diet on their protein metabolism, 18 3-month-old Sunit sheep were randomly divided into two groups (Group C (non-feeding of LAB) and Group L (feeding of LAB)) for a 90 d feeding trial. After the animals were slaughtered, we measured several key factors, including basal indices, amino acid composition, protein metabolism enzyme activity, and protein metabolism-related gene expression. There were nine replications of each group. The data show that LAB had a significant effect on the intramuscular fat (IMF) content (P<0.001). The composition of amino acids in the serum and mutton was improved by the addition of LAB. The addition of LAB led to a significant increase in the expression of genes related to AKT serine/threonine kinase 1 (Akt1), mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K1), and eukaryotic initiation factor 4E (eIF4E) protein synthesis (P<0.05), while the expression of all genes related to protein catabolism was reduced. Thus, dietary LAB can increase IMF content, improve amino acid composition, regulate protein metabolism in lambs, and provide a new way and theoretical basis for dietary management to improve meat quality in housed sheep.

Discovery of potential RSK1 inhibitors for cancer therapy using virtual screening, molecular docking, molecular dynamics simulation, and MM/GBSA calculations

The p90 ribosomal protein S6 Kinase (RSK) family belongs to Ser/Thr protein kinases that includes four isoform RSK1-4 in mammals. The ribosomal protein S6 Kinase 1 (RSK1) is also known as ribosomal protein S6 kinase alpha-1 (RPS6KA1) is a special protein due to their two catalytic regions that is associated with abundantly various cancers and it is proposed as a drug target. Several RSK1 isoform inhibitors have been reported but none of them are used in clinical studies. Thus, we aimed to perform ligand pharmacophore mapping with the known inhibitor and structure-based virtual screening studies to determine potential candidates against RSK1-terminal kinase domains CTKD and NTKD. The studied compounds from the databases (ApexBio, ChEMBL, ChemDiv). The molecular docking study was performed with the resulted candidates by using CDOCKER and Glide/SP methods. The four candidates with the highest docking scores were used for further 100-ns molecular dynamics (MD) simulations and Molecular Mechanics Generalised Born and Surface Area (MM/GBSA) calculations. The root mean square deviation (RMSD) for protein complexes were found between 2 Å and 4 Å. Solvent accessible surface area (SASA), radius of gyration (Rg), and polar surface area (PSA) values were calculated for compounds. The binding free energies were calculated between −72.22 kcal/mol and −82.44 kcal/mol. The interaction diagrams showed that hydrogen bond, alkyl, and π-alkyl interactions were observed with specific residues such as Leu144, Lys94, Asp142 for RSK1-NTKD, and Cys532, Cys556, Lys447, Asn540 for RSK1-CTKD. The identified compounds may be potential inhibitor candidates of RSK1 following the preclinical studies. Communicated by Ramaswamy H. Sarma

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.

Vitamin B12-Induced Autophagy Alleviates High Glucose-Mediated Apoptosis of Islet β Cells.

High glucose levels can lead to the apoptosis of islet β cells, while autophagy can provide cytoprotection and promote autophagic cell death. Vitamin B12, a water-soluble B vitamin, has been shown to regulate insulin secretion and increase insulin sensitivity. However, the precise mechanism of action remains unclear. In this study, we investigated the influence of vitamin B12 on high glucose-induced apoptosis and autophagy in RIN-m5F cells to elucidate how vitamin B12 modulates insulin release. Our results demonstrate that exposure to 45 mM glucose led to a significant increase in the apoptosis rate of RIN-m5F cells. The treatment with vitamin B12 reduced the apoptosis rate and increased the number of autophagosomes. Moreover, vitamin B12 increased the ratio of microtubule-associated protein 1 light chain 3 beta to microtubule-associated protein 1 light chain 3 alpha (LC3-II/LC3-I), while decreasing the amount of sequestosome 1 (p62) and inhibiting the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K) under both normal- and high-glucose conditions. The additional experiments revealed that vitamin B12 inhibited high glucose-induced apoptosis. Notably, this protective effect was attenuated when the autophagy inhibitor 3-methyladenine was introduced. Our findings suggest that vitamin B12 protects islet β cells against apoptosis induced by high glucose levels, possibly by inducing autophagy.

Resveratrol improves palmitic acid‑induced insulin resistance via the DDIT4/mTOR pathway in C2C12 cells.

The present study aimed to establish a model of palmitic acid (PA)‑induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage‑inducible transcript 4 (DDIT4)‑small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription‑quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)‑1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)‑IRS‑1, p‑mTOR and p‑p70S6K protein expression levels were increased, and p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were increased, p‑IRS‑1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4‑siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4‑siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p‑PI3K, p‑AKT and GLUT4 protein expression levels were down‑regulated, p‑IRS‑1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA‑induced IR in C2C12 cells through the DDIT4/mTOR/IRS‑1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.

Effects of Dietary Lysine Level on Growth Performance and Protein Metabolism in Juvenile Leopard Coral Grouper (Plectropomus leopardus).

An 8-week feeding trial was conducted to evaluate the effects of dietary lysine level on growth performance and protein metabolism of juvenile leopard coral grouper (Plectropomus leopardus) and thereby obtained the optimal dietary lysine requirement of P. leopardus. Six isoproteic and isolipidic experimental diets were formulated to contain 1.10%, 1.69%, 2.30%, 3.08%, 3.56%, and 4.36% lysine of diets, respectively. Each diet was assigned at random to triplicate groups of 25 juveniles (initial mean weight is 10.57 g) per tank in a flow-through mariculture system maintained at 27-30°C. Dietary inclusion of 2.30-3.08% lysine improved the weight gain rate (WGR) and specific growth rate and decreased the feed conversion ratio (FCR) of juveniles (P < 0.05). The intestinal digestive enzyme (trypsin, amylase, and lipase) activities were overall enhanced by dietary inclusion of 3.08-3.56% lysine (P < 0.05). The mammalian target of rapamycin (mTOR) signaling pathway was activated in fish fed diets with 1.69-2.30% lysine by upregulating the relative expression levels of hepatic TOR and S6K1 (p70 ribosomal protein S6 kinase 1) but downregulating the relative expression level of hepatic 4E-BP2 (eIF4E-binding protein 2). Conversely, the amino acid response signaling pathway was inhibited in fish fed diet with 2.30% lysine by downregulating the relative expression levels of hepatic GCN2 (general control nondepressible 2), ATF3 (activating transcription factor 3), ATF4a (activating transcription factor 4a), and ATF4b (activating transcription factor 4b). Additionally, dietary 1.69-3.08% lysine enhanced the plasma total protein level and hepatic lysine α-ketoglutarate reductase activity but depressed the blood urea nitrogen level and hepatic adenosine monophosphate deaminase activity (P < 0.05). Moreover, dietary 3.08% lysine increased the contents of whole-body crude protein and total amino acids, while 1.69%-4.36% lysine depressed the whole-body lipid content (P < 0.05). These results indicated that optimal dietary lysine increased the digestive enzyme activities, promoted protein synthesis but depressed protein degradation, and thereby improved the growth performance of P. leopardus. Based on the second-order polynomial model, the optimal lysine requirement of juvenile P. leopardus for WGR, FCR, and lysine deposition was 2.60%-2.97% of diets (4.91%-5.60% of dietary protein).

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

Effects of L-Arginine and Arginine-Arginine dipeptide on amino acids uptake and αS1-casein synthesis in bovine mammary epithelial cells.

Arginine (Arg), as an important functional amino acids (AA), is essential for milk protein synthesis in lactating ruminants. Arg shares transporters with cationic and neutral AA in mammary epithelial cells. Therefore, competitive inhibition might exist among these AA in uptake by mammary epithelial cells. In this study, cultured bovine mammary epithelial cells (BMEC) was used as the model to investigate whether the availability of L-Arg (0.7, 1.4, 2.8, 5.6 and 11.2mM) affects the uptake of other AA and if this related to αS1-casein synthesis, and whether Arginine-Arginine (Arg-Arg) substituting part of free L-Arg can alleviate competitive inhibition among Arg and other AA, so as to promote αS1-casein synthesis. Our results showed 2.8mM L-Arg generated the greatest positive effects on αS1-casein synthesis and the activation of mammalian target of rapamycin (mTOR) signaling pathway (P < 0.01). With L-Arg supply increasing from 0.7 to 11.2mM, the net-uptake of other AA (except Glu and Ala) decreased linearly and quadratically (Plinear < 0.01; Pquadratic < 0.01). Compared with 2.8mM, the net-uptake of essential amino acids (EAA) and total amino acids (TAA) were lower at 11.2mM L-Arg group, while greater at 1.4mM L-Arg group (P < 0.01). Arg-Arg dipeptide replacing 10% free L-Arg increased αS1-casein synthesis (P < 0.05), net-uptake of EAA and TAA, as well as phosphorylation level of mTOR and p70 ribosomal protein S6 kinase (P70S6K) and mRNA expression of oligopeptide transporter 2 (PepT2; P < 0.01). These observations suggested that the increased αS1-casein synthesis by 10% Arg-Arg dipeptide might be related to the increase of AA availability and the activation of mTOR signaling pathway in BMEC.

Vitamin B6 Inhibits High Glucose-Induced Islet β Cell Apoptosis by Upregulating Autophagy.

Vitamin B6 may alleviate diabetes by regulating insulin secretion and increasing insulin sensitivity, but its mechanism remains to be explored. In this study, vitamin B6-mediated autophagy and high glucose-induced apoptosis were tested to investigate the mechanism by which vitamin B6 regulates insulin release. The results showed that 20 mM glucose increased the apoptosis rate from 10.39% to 22.44%. Vitamin B6 reduced the apoptosis rate of RIN-m5F cells from 22.44% to 11.31%. Our data also showed that the vitamin B6 content in processed eggs was decreased and that the hydrothermal process did not affect the bioactivity of vitamin B6. Vitamin B6 increased the number of autophagosomes and the ratio of autophagosome marker protein microtubule associated protein 1 light chain 3 beta to microtubule associated protein 1 light chain 3 alpha (LC3-II/LC3-I). It also decreased the amount of sequetosome 1 (SQSTM1/p62) and inhibited the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K) under normal and high glucose stress. Another study showed that vitamin B6 inhibited the apoptosis rate, whereas the autophagy inhibitor 3-methyladenine (3-MA) blocked the protective effect of vitamin B6 against apoptosis induced by high glucose. The hydrothermal process decreased the vitamin B6 content in eggs but had no effect on the cytoprotective function of vitamin B6 in RIN-m5f cells. In conclusion, we demonstrated that vitamin B6-mediated autophagy protected RIN-m5f cells from high glucose-induced apoptosis might via the mTOR-dependent pathway. Our data also suggest that low temperatures and short-term hydrothermal processes are beneficial for dietary eggs.

Effects of low-dose photodynamic therapy on the function of human adipose mesenchymal stem cells and its mechanism

Objective: To investigate the effects of low-dose photodynamic therapy on the proliferation, regulation, and secretion functions of human adipose mesenchymal stem cells (ADSCs) and the related mechanism, so as to explore a new method for the repair of chronic wounds. Methods: The experimental research methods were adopted. From February to April 2021, 10 patients (5 males and 5 females, aged 23 to 47 years) who underwent cutaneous surgery in the Department of Dermatology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) donated postoperative waste adipose tissue. The cells were extracted from the adipose tissue and the phenotype was identified. Three batches of ADSCs were taken, with each batch of cells being divided into normal control group with conventional culture only, photosensitizer alone group with conventional culture after being treated with Hemoporfin, irradiation alone group with conventional culture after being treated with red light irradiation, and photosensitizer+irradiation group with conventional culture after being treated with Hemoporfin and red light irradiation, with sample number of 3 in each group. At culture hour of 24 after the treatment of the first and second batches of cells, the ADSC proliferation level was evaluated by 5-ethynyl-2'-deoxyuridine staining method and the migration percentage of HaCaT cells cocultured with ADSCs was detected by Transwell experiment, respectively. On culture day of 7 after the treatment of the third batch of cells, the extracellular matrix protein expression of ADSCs was detected by immunofluorescence method. The ADSCs were divided into 0 min post-photodynamic therapy group, 15 min post-photodynamic therapy group, 30 min post-photodynamic therapy group, and 60 min post-photodynamic therapy group, with 3 wells in each group. Western blotting was used to detect the protein expressions and calculate the phosphorylated mammalian target of rapamycin complex (p-mTOR)/mammalian target of rapamycin (mTOR), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K)/p70 ribosomal protein S6 kinase (p70 S6K) ratio at the corresponding time points after photodynamic therapy. Two batches of ADSCs were taken, and each batch was divided into normal control group, photodynamic therapy alone group, and photodynamic therapy+rapamycin group, with 3 wells in each group. At culture minute of 15 after the treatment, p-mTOR/mTOR and p-p70 S6K/p70 S6K ratios of cells from the first batch were calculated and detected as before. On culture day of 7 after the treatment, extracellular matrix protein expression of cells from the second batch was detected as before. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 12 d of culture, the cells were verified as ADSCs. At culture hour of 24 after the treatment, the ADSC proliferation level ((4.0±1.0)% and (4.1±0.4)%, respectively) and HaCaT cell migration percentages (1.17±0.14 and 1.13±0.12, respectively) in photosensitizer alone group and irradiation alone group were similar to those of normal control group ((3.7±0.6)% and 1.00±0.16, respectively, P>0.05), and were significantly lower than those of photosensitizer+irradiation group ((34.2±7.0)% and 2.55±0.13, respectively, P<0.01). On culture day of 7 after the treatment, compared with those in normal control group, the expression of collagen Ⅲ in ADSCs of photosensitizer alone group was significantly increased (P<0.05), and the expressions of collagen Ⅰ and collagen Ⅲ in ADSCs of irradiation alone group were significantly increased (P<0.01). Compared with those in photosensitizer alone group and irradiation alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photosensitizer+irradiation group were significantly increased (P<0.01). Compared with those in 0 min post-photodynamic therapy group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in 15 min post-photodynamic therapy group were significantly increased (P<0.01), the ratios of p-p70 S6K/p70 S6K of ADSCs in 30 min post-photodynamic therapy group and 60 min post-photodynamic therapy group were both significantly increased (P<0.01). At culture minute of 15 after the treatment, compared with those in normal control group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy alone group were significantly increased (P<0.05 or P<0.01). Compared with those in photodynamic therapy alone group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.05). On culture day of 7 after the treatment, compared with those in normal control group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy alone group were significantly increased (P<0.01). Compared with those in photodynamic therapy alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.01). Conclusions: Low-dose photodynamic therapy can promote the proliferation of ADSCs, improve the ability of ADSCs to regulate the migration of HaCaT cells, and enhance the secretion of extracellular matrix protein by rapidly activating mTOR signaling pathway.

Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway

To investigate the effect of moxibustion on autophagy and amyloid β-peptide1-42 (Aβ1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aβ1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

The effect of high-intensity interval training (HIIT) on protein expression in Flexor Hallucis Longus (FHL) and soleus (SOL) in rats with type 2 diabetes.

In people with diabetes, one of the problems for patients is muscle wasting and inhibition of the protein synthesis pathway. This study aimed to evaluate the effects of HIIT on protein expression in two skeletal muscles, flexor hallucis longus (FHL) and soleus (SOL) in rats with type 2 diabetes mellitus (T2DM). Diabetes initially was induced by streptozotocin (STZ) and nicotinamide. Rats with type 2 diabetes were randomly and equally divided into control (n = 6) and HIIT groups (n = 6). After 8 weeks of training, the content of total and phosphorylated proteins of serine/threonine-protein kinases (AKT1), mammalian target of rapamycin (mTOR), P70 ribosomal protein S6 kinase 1 (P70S6K1), and 4E (eIF4E)-binding protein 1 (4E-BP1) in FHL and SOL muscles were measured by Western blotting. While body weight and blood glucose were also controlled. In the HIIT training group, compared to the control group, a significant increase in the content of AKT1 (0.003) and mTOR (0.001) proteins was observed in the FHL muscle. Also, after 8 weeks of HIIT training, protein 4E-BP1 (0.001) was increased in SOL muscle. However, there was no significant change in other proteins in FHL and SOL muscle. In rats with type 2 diabetes appear to HIIT leading to more protein expression of fast-twitch muscles than slow-twitch muscles. thus likely HIIT exercises can be an important approach to increase protein synthesis and prevent muscle atrophy in people with type 2 diabetes.

Ribosomal S6 Protein Kinase 2 Aggravates the Process of Systemic Scleroderma

Systemic sclerosis is a complex process of pathogenesis, and the contributions of inherited genes, infections, and chemicals remain largely unknown. In this study, we showed that p90 ribosomal S6 protein kinase 2 (RSK2) was selectively upregulated in fibrotic skin and fibroblasts treated with the profibrotic cytokine TGF-β. Moreover, knockout of Rsk2 specifically in skin fibroblasts or pharmacological inhibition of RSK2 attenuated skin fibrosis in a mouse model. Mechanistically, RSK2 directly interacted with glycogen synthase kinase 3β invivo and invitro and thereby induced phosphorylation of glycogen synthase kinase 3β at Ser9 to inhibit ubiquitination and degradation of GLI1, which promoted fibroblast differentiation and skin fibrosis. Consequently, RSK2 plays an important role in the dermal skin of systemic sclerosis. These findings provided a potential therapeutic target for systemic sclerosis.

Unraveling the therapeutic potential of GANT61/Dactolisib combination as a novel prostate cancer modality

Aberrant activation of several signaling pathways has been implicated in prostate cancer (PCa) progression to castrate-resistant prostate cancer (CRPC). Phosphoinositide-3-kinase/Protein Kinase B/mechanistic Target of Rapamycin (PI3K/AKT/mTOR) and Hedgehog/GLI (Hh/GLI) pathways are major participants in progression to CRPC. In this sense, the current work aims to assess the potential antitumor effects resulting from co-targeting the aforementioned pathways in PC3 cells with Dactolisib as a dual PI3K/mTOR inhibitor and GANT61 as a GLI1 antagonist. Three replica of PC3 cells were assigned for four treatment groups; vehicle control, Dactolisib-treated, GANT61-treated, and combination-treated groups. GLI1 gene expression was determined by quantitative real-time PCR while active caspase-3 was determined colorimetrically. P-AKT, p70 ribosomal s6 protein kinase 1 (pS6K1), cyclin D1, vascular endothelial growth factor 1 (VEGF1), and Microtubule-associated proteins 1A/1B light chain 3 (LC3) protein levels were determined by ELISA technique. GLI1 gene expression was down-regulated as a result of Dactolisib, GANT61, and their combination. Additionally, both drugs significantly reduced p-AKT, pS6K1, cyclin D1, and VEGF1 protein levels. Dactolisib elevated LC3 protein levels and GANT61 augmented Dactolisib effect on LC3. Moreover, only Dactolisib/GANT61combination significantly increased active caspase-3 level. To sum up, Dactolisib/GANT61 combination was shown to be promising in PCa treatment. Further in-vitro and in-vivo studies are warranted to support our findings.

L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathway

Milk production determines health status and optimal growth of suckling neonates. Recent studies revealed that the secretion of leucine into sow milk from the mammary gland is significantly lower than the amount taken up by the mammary gland from the blood, suggesting the potential and fundamental role in udder metabolism. However, little is known about the underlying mechanism. In this study, porcine mammary epithelial cells (PMECs) were employed to investigate the effects of leucine on protein synthesis and mammalian target of rapamycin (mTOR) signalling. Results showed that leucine supplementation significantly increased the proliferation of PMECs in a dose-dependent manner from 0 to 1 mmol/l. Secretions of four casein subunits were significantly increased by addition of leucine. The phosphorylation of proteins related to mTOR signalling, including mTOR, eukaryotic translation initiation factor 4E-binding protein, and p70 ribosomal protein S6 kinase, were accordingly upregulated by leucine supplementation. Casein secretion was not affected by leucine treatment when mTOR signalling was inhibited by rapamycin. This suggests that leucine stimulates protein synthesis through the mTOR signalling pathway. These findings facilitate the utilization of dietary leucine supplementation to improve milk production in lactating sows.

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.