mTOR Protein Levels Research Articles

Role of autophagy in lipopolysaccharide-induced apoptosis of odontoblasts

To investigate the role of autophagy in lipopolysaccharide (LPS)-induced apoptosis of murine odontoblasts. Murine odontoblasts (mDPC-23 cells) were treated with 5 μg/mL LPS for 6, 12 and 24 h, and the changes in cell viability was examined using CCK8 kit and cell apoptosis was detected by TUNEL staining. The changes in the protein levels of LC3, Beclin1, Atg5, AKT, p-AKT, mTOR and p-mTOR were detected using Western blotting. The effect of 3-MA treatment for 24 h on LPS-induced apoptosis of mDPC-23 cells was evaluated by detecting the expressions of apoptosis-related proteins caspase-3 and Bax using Western blotting. Stimulation with LPS for 6 and 12 h did not cause significant changes in the proliferation or apoptosis of mDPC-23 cells, but LPS treatment for 24 h significantly suppressed cell proliferation (P < 0.05) and promoted cell apoptosis as shown by TUNEL assay (P < 0.05). Stimulation with LPS for 24 significantly increased the expression levels of LC3, Beclin1 and Atg5, decreased the expressions of p-AKT and p-mTOR (P < 0.05), and obviously upregulated the expressions of caspase-3 and Bax (P < 0.05). Treatment with 3-MA markedly lowered caspase-3 and Bax protein expressions in LPS-stimulated cells (P < 0.05). LPS stimulation induces autophagy to promote apoptosis of mDPC-23 cells, and suppression of autophagy attenuates LPS-induced apoptosis. Autophagy may play an important role in the injury of inflamed pulp tissues.

The endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase G pathway activates primordial follicles

In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.

The mechanism study of ouabain in inhibiting the growth of hepatocellular carcinoma cells by inhibiting the laser kinase signaling pathway

Objective: To investigate the effect of Na(+)/K(+)-ATPase inhibitor ouabain on the proliferation and division of liver cancer HepG2 cells, and to explore the anticancer mechanism. Methods: HepG2 cells were exposed with different concentrations of ouabain (0.1, 1, 10 μmol/L) for 24 h, the proliferation ability was appraised using CCK-8, and the HepG2 cells was as a control group. The status of chromosome separation was detected with cell immunofluorescence (ICC) coupled to confocal microscope. The expression levels of AURKA, mTOR, p-mTOR, ERK and p-ERK protein were analyzed using western blot. Results: After treating with 0.1, 1 and 10 μmol/L of ouabain for 24 h, the inhibitory rate of cells were (23.5±4.57)%, (49.80±5.32)%, and (72.10±5.62)%, respectively. Ouabain could significantly inhibit the proliferation of HepG2, and presented in a dose-dependent manner(F=32.8, P<0.05). The ICC results showed that the chromosome separation disorders occurred in HepG2 cells treated with 1 μmol/L for 24 h, and the spindle diameter of HepG2 cells with ouabain treatment was decreased significantly compared with the control group(t=9.58, P<0.05). The results of western blot showed that the expression levels of AURKA, p-mTOR and p-ERK expressions in HepG2 cells treated with 1 μmol/L of ouabain were significantly decreased compared with the control group(F=16.26, 8.32, 33.59, P<0.05). Ouabain inhibited the growth of hepatocellular carcinoma cells in nude mice(F=370.20, P<0.05). Conclusion: Ouabain can induce chromosome division disorder and inhibit the proliferation in liver cancer HepG2 cells by inhibiting AURKA signaling pathway.

Social context-dependent singing alters molecular markers of synaptic plasticity signaling in finch basal ganglia Area X

Vocal communication is a crucial skill required throughout life. However, there is a critical gap in our understanding of the underlying molecular brain mechanisms, thereby motivating our use of the zebra finch songbird model. Adult male zebra finches show differences in neural activity patterns in song-dedicated brain nuclei when they sing in two distinct social contexts: a male singing by himself (undirected, UD) and a male singing to a female (female-directed, FD). In our prior work, we showed that in song-dedicated basal ganglia Area X, protein levels of a N-methyl-D-aspartate receptor subtype 2B (NMDAR2B) increased with more UD song and decreased with more FD song. We hypothesized that molecules downstream of this receptor would show differential protein expression levels in Area X between UD and FD song. Specifically, we investigated calcium/calmodulin dependent protein kinase II beta (CaMKIIB), homer scaffold protein 1 (HOMER1), serine/threonine protein kinase (Akt), and mechanistic target of rapamycin kinase (mTOR) following singing and non-singing states in Area X. We show relationships between social context and protein levels. HOMER1 protein levels decreased with time spent singing FD song, and mTOR protein levels decreased with the amount of and time spent singing FD song. For both HOMER1 and mTOR, there were no differences with the amount of UD song. With time spent singing UD, CaMKIIB protein levels trended in a U-shaped curve whereas Akt protein levels trended down. Both molecules showed no change with FD song. Our results support differential involvement of molecules in synaptic plasticity pathways between UD and FD song behaviors.

Mitochondrial Energy-Regulating Effect of Atractyloside Inhibits Hepatocellular Steatosis Through the Activation of Autophagy.

Background and AimAtractyloside (ATR), a mitochondrial uncoupler, is known for its specific inhibition of mitochondrial oxidative phosphorylation. Previous studies have reported that moderate mitochondrial uncoupling effect is beneficial to increase the decomposition and clearance of hepatic lipid, prevent the occurrence of fatty liver diseases. Moreover, the beneficial effects of mitochondrial uncouplers on type 2 diabetes and metabolic syndromes have been consistently observed. The present study investigated the effect of ATR on steatosis level of HepG2 cells treated with free fatty acid (FFA).MethodsIntracellular triglyceride level and Oil Red O staining were assessed, the mitochondrial adaptation and ADP/ATP ratio were analyzed, the protein level of AMPK, mTOR and LC3B, autophagic flux, and the co-localization of LC3B with lipid droplets was performed.ResultsATR treatment inhibited the activity of mitochondrial respiratory chain complexes I and IV, decreased the mitochondrial membrane potential, and increased the ADP/ATP ratio in the FFA-treated cells. Furthermore, ATR increased the gene expression and protein level of LC3B and promoted the autophagic flux processing from early autophagosome to late autolysosome by increasing the protein level of AMPKα and decreasing the protein level of mTOR. An increased number of autophagosomes (LC3B) was also observed in the lipid droplets. ATR treatment accelerated lipid degradation in the FFA-treated cells, and the lowest lipid content was observed in the cell group with 7.5 μM ATR.ConclusionLow concentrations (2.5, 5, and 7.5 μM) of ATR treatment could activate autophagy to accelerate the degradation of TGs in steatosis HepG2 cells; the mechanism may be related to the activation of the AMPK/mTOR pathway induced by the increased ADP/ATP ratio. In addition, the ideal concentration of ATR for improving steatotic HepG2 cells was 7.5 μM.

Maternal low-protein diet on the last week of pregnancy contributes to insulin resistance and β-cell dysfunction in the mouse offspring.

Maternal low-protein diet (LP) throughout gestation affects pancreatic β-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for β-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal β-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with β-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient β-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

An Analysis of Free Plasma Amino Acid Profile and mTOR Protein Level in Children with Malnutrition

Amaç: Malnütrisyon bir ya da daha fazla besin öğesinin vücut dengesini bozacak şekilde yetersiz alınması sonucunda ortaya çıkan klinik bir tablodur. Etiyopatogenezin de beslenme problemleri, protein ve enerji eksikliği ve özellikle mTOR proteini ve kan aminoasitleri ile ilgili eksiklikler mevcuttur. Bu çalışmada büyüme ve gelişme geriliği olan çocukların serbest plazma aminoasit profili ve mTOR protein düzeyinin araştırılmasını amaçladık. Materyal ve metod: Çalışmamızda hastanemiz pediatri polikliniğine başvuran 0-18 yaş arasında boy ve kilosu üç persantilin altında olan malnütrisyonlu çocuklarda mTOR düzeyi ve mTOR düzeyinin kan aminoasit düzeyleriyle ilişkisi incelenmiştir. Bulgular: Çalışmaya 48 hasta ve kontrol grubu için tamamen sağlıklı 47 çocuk alındı. Beslenmenin yetersizliğine bağlı olarak malnütrisyon olan çocuklarda mTOR ve aminoasit seviyelerinde düşüklük beklenmesine rağmen referans aralıklarına göre değerlendirildiğinde kontrol grubu ile hasta grubu arasında anlamlı fark bulunamadı. Sonuç: Büyüme ve gelişme geriliği olan çocuklarda bakılan mTOR ve aminoasit profili özel bir belirteç olarak kullanılamasa da bu alanda yapılacak birçok çalışmaya ışık tutabileceği kanısına varıldı.

Dichloromethane extraction from Piper nigrum L. and P. longum L. to mitigate ischemic stroke by activating the AKT/mTOR signaling pathway to suppress autophagy

Dichloromethane fraction (DF) of Piper nigrum L. and P. longum L. (PnL and PlL), has been found to exert a protective effect against ischemic stroke in rats. However, the regulatory mechanism exerted by PnL and PIL have not been fully elucidated. In this study, we found that DF greatly ameliorated cerebral ischemic injury in a rat model of permanent middle cerebral artery occlusion (pMCAO). The neurological score, behavioral assessment, brain infarct volume, phosphorylation of AKT (p-AKT), phosphorylation mTOR (p-mTOR), and Atg7 protein levels were determined. Additionally, we discovered that DF pretreatment reduced infarct volume, neurological score, and brain damage. Furthermore, DF therapy caused the downregulation of Atg7 and p-AKT expression, as well as the upregulation of p-mTOR expression. In conclusion, our findings indicated that DF treatment can reduce brain damage and inhibit apoptosis and autophagy by activating the Akt-mTOR signaling pathway in ischemic stroke.

Implications of miRNAs on TGF-β/TAK1/mTOR pathway in mediating the renoprotective effects of pentoxifylline against cisplatin-induced nephrotoxicity in rats

Cisplatin (CIS)-mediated nephrotoxicity is induced via transforming growth factor-beta (TGF-β) and TGF-β-activated kinase (TAK1). TGF-β and TAK1 are known to interact with microRNA-let-7b and microRNA-26b, respectively. Additionally, TGF-β1 is reported to down-regulate the autophagy marker microtubule-associated protein 1 light chain 3-II (LC3-II) through upregulation of microRNA-34a. Pentoxifylline (PTX) anti-inflammatory effects are mediated via suppressing TGF-β and regulating mammalian target of rapamycin (mTOR).The current study aimed to investigate the involvement of microRNAs let-7b, 26b, and 34a, and the modulating impact of PTX on CIS-induced nephrotoxicity. Moreover, we aimed at examining the ability of PTX to interact with TGF-β receptor-1 (TGFβR-1), and TAK1, and examine its ability to downgrade the previously reported toxicities. Hence, the expression of the aforementioned microRNAs, and protein levels of TGFβR-1, TGF-β1, TAK1, mTOR, LC3-II, and NF-κB were assessed. Molecular docking studies of PTX on TGFβR-1 and TAK1 were also executed.CIS induced TGF-β1, with down-regulation of microRNA-let-7b and -26b, and up-regulation of microRNA-34a. TGFβR-1, TAK1, and mTOR levels were increased, while LC3-II level was decreased. PTX significantly protected renal cells against CIS-induced changes as indicated by reverting the level of the investigated parameters, while exhibiting an antagonistic effect on TGFβR-1 and TAK1.Our results postulate a possible role of epigenetic regulation of CIS-induced nephrotoxicity through the investigated microRNAs proposing them as potential future targets for controlling this serious toxicity. PTX was able to shield CIS-induced toxicity possibly through blocking TGF-β pathway, while promoting autophagy in a TAK1 independent manner with the involvement of the examined microRNAs.

Effects of forced swimming stress on expression and phosphorylation of PI3K/Akt signal pathway in pancreas of type 2 diabetic rats.

BackgroundTo study the effects of forced swimming stress on blood glucose and insulin levels and the expression and phosphorylation of pancreatic PI3K/Akt signal pathway in type 2 diabetic rats.MethodsThirty adult SD rats (8-week-old, male) were randomly divided into three groups: control group, diabetic model group, and diabetic model stress group. The diabetic model group was established by feeding with the high-fat and high-glucose diet for four weeks, and then the rats were injected with low-dose streptozotocin (STZ, 25 mg/kg, once a day for 2 days). The rats in the diabetic model group were subjected to forced swimming stress for seven days, which was a diabetic model stress group. Twenty-four hours after the last forced swimming stress, the rats fasted, and blood samples were collected to detect blood glucose, insulin, triglycerides (TGs), free fatty acids (FFAs), high- and low-density lipoprotein cholesterol (HDLC and LDLC) levels. The western blot was applied to detect the expression of PI3K, Akt, p-Akt, and mTOR in the pancreas of rats in each group.ResultsBlood glucose and insulin levels in the diabetic model stress group were significantly lower than those in the diabetic model group, and the protein levels of PI3K, p-AKT, and mTOR in the pancreas of the diabetic model stress group were significantly higher than those of the diabetic model group.ConclusionsOne-week swimming stress can decrease the blood glucose level and improve insulin indexes in type 2 diabetic rats and increase the expression and phosphorylation of pancreatic PI3K/Akt signal pathway proteins.

Artesunate ameliorates sepsis-induced acute lung injury by activating the mTOR/AKT/PI3K axis.

Sepsis-induced acute lung injury (ALI) remains one of the most common disorders in hospitals. The aim of this research was to explore the underlying characteristics and mechanisms of artesunate (AS) in protecting rat lungs from sepsis. The sepsis-induced ALI model was generated in SD rats by the intratracheal administration of lipopolysaccharide (LPS, 5mg/kg) for 24h. The rats were randomly assigned into 4 groups: Sham, LPS, LPS+AS, and LPS+AS+LY294002. Pathological evaluation of the lungs was conducted by HE staining, immunofluorescence, and TUNEL assay, and the MPO activity and the W/D ratio of each group were evaluated. The levels of inflammatory mediators (TNF-α and IL-6) were measured by immunoblotting, immunofluorescence and real-time PCR. Western blotting was used to determine the protein levels of PI3K, cleaved Caspase-3, p-mTOR, mTOR, p-Akt, and Akt in the lungs. The MPO activity, W/D ratio and lung injury score were obviously elevated after induction of ALI by LPS. Nevertheless, these alterations could be inhibited by AS. In addition, sepsis decreased the levels of p-mTOR, p-Akt, and PI3K and elevated the expression of cl-caspase-3, TNF-α, and IL-6 in the lungs. After AS administration, these alterations were obviously decreased, but treatment with the PI3K antagonist LY294002 inhibited the function of AS. AS could partially protect against LPS-induced ALI by inhibiting apoptosis and inflammatory mediator production, and this function is perhaps associated with the regulation of the mTOR/AKT/PI3K axis. These findings suggest that AS may possess certain beneficial characteristics in protecting the lungs from sepsis.

MicroRNA-99a Restricts Replication of Hepatitis C Virus by Targeting mTOR and de novo Lipogenesis.

In this study, we investigated the role of microRNA-99a (miR-99a) in hepatitis C virus (HCV) replication and lipogenesis in hepatocytes. Cell-culture-derived HCV (HCVcc) infection caused down-regulation of miR-99a in Huh-7 cells, and the relative levels of miR-99a were significantly lower in the sera of the HCV-infected patients than in those of healthy controls. Transfection of miR-99a-5p mimics resulted in a decrease in the intracellular and secreted HCV RNA levels. It also caused a decreased mammalian target of rapamycin (mTOR) protein level and phosphorylation of its downstream targets in HCV-replicating cells. Sterol regulatory element binding protein (SREBP)-1c expression and intracellular lipid accumulation decreased when either miR-99a-5p mimics or si-mTOR was transfected in oleic acid-treated Huh-7 cells. Overexpression of mTOR rescued HCV RNA replication and lipid droplet accumulation in miR-99a-5p mimics-transfected HCV replicon cells. Our data demonstrated that miR-99a ameliorates intracellular lipid accumulation by regulating mTOR/SREBP-1c and causes inefficient replication and packaging of intracellular HCV.

Differentiation proliferative capacity of skeletal muscle satellite cells from Dapulian and Landrace pigs

Previously, we found there were significant differences in the development of skeletal muscle, myofiber characteristics between Chinese pig breed Dapulian and Landrace. Furthermore, the development of skeletal muscle is related to the proliferation of satellite cells (SCs). Thus, our aim was to compare the differences of proliferative abilities and expression of key genes related to proliferation between the SCs isolated from Dapulian and Landrace in this study. We isolated skeletal muscle SCs with collagenase digestion from the longissimus dorsi muscles of 1-day-old male Dapulian and Landrace piglets. After successful culture of SCs in vitro, proliferative capacity was estimated at different time points. Meanwhile, the expression of key genes related to SCs proliferation was detected by qRT-PCR and western blotting. Proliferation results showed a significantly greater number of viable SCs from Dapulian pigs compared to that from Landrace pigs at 72–120 h of culture (p < .05). The mRNA expression of MYOD, MYF5 and MYOG in SCs from Dapulian pigs was higher (p < .05) compared with Landrace pigs. Whereas the mRNA levels of MSTN, MTOR, S6K, EIF4E and 4EBP1 were significantly lower in SCs from Dapulian than those from Landrace (p < .05). Protein levels of MSTN, S6K, MTOR and EIF4E were consistent with corresponding mRNA expression. In summary, these findings indicated that SCs isolated from the two breeds exhibit different proliferative capacities, and the proliferative potential of SCs from Dapulian pigs was greater than those from Landrace pigs. Highlights There were significant differences in the development of skeletal muscle, myofibre number and myofibre size between Chinese indigenous pig breed Dapulian and commercial pig breed Landrace. The proliferative capacity of satellite cells (SCs) from Dapulian pigs was greater than that from Landrace pigs. The expression of key genes related to proliferation between the SCs isolated from Dapulian and Landrace was different.

Upregulation of miR-101 during Influenza A Virus Infection Abrogates Viral Life Cycle by Targeting mTOR Pathway.

Micro RNAs (miRNAs) are a class of small non-coding single-stranded RNA, which play an important role in modulating host-Influenza A virus (IAV) crosstalk. The interplay between influenza and miRNA interaction is defined by a plethora of complex mechanisms, which are not fully understood yet. Here, we demonstrate that in IAV infected A549 cells, a synchronous increase was observed in the expression of mTOR up to 24 hpi and significant downregulation at 48 hpi. Additionally, NP of IAV interacts with mTOR and modulates the levels of mTOR mRNA and protein, thus regulating the translation of host cell. RNA sequencing and qPCR analysis of IAV-infected A549 cells and NP transfected cells revealed that miR-101 downregulates mTOR transcripts at later stages of infection. Ectopic expression of miR-101 mimic led to a decrease in expression of NP, a reduction in IAV titer and replication. Moreover, treatment of the cells with Everolimus, a potent inhibitor of mTOR, resulted in an increase of miR-101 transcript levels, which further suppressed the viral protein synthesis. Collectively, the data suggest a novel mechanism that IAV stimulates mTOR pathway at early stages of infection; however, at a later time-point, positive regulation of miR-101 restrains the mTOR expression, and hence, the viral propagation.

Baicalin alleviates deoxynivalenol-induced intestinal inflammation and oxidative stress damage by inhibiting NF-κB and increasing mTOR signaling pathways in piglets

This study was conducted to evaluate the intestinal protective effects of baicalin (BAI) in deoxynivalenol (DON)-treated piglets. A total of 320 weaned piglets were randomly allotted to 1 of 4 treatments with 8 replication pens per treatment and 10 piglets per pen. The treatments were basal diet (NC), basal diet + 0.1% baicalin (BAI), basal diet + 4 mg/kg DON (DON), and basal diet + 4 mg/kg DON + 0.1% BAI (DON + BAI). The experiment was conducted for 14 days. BAI supplementation alleviated the impairment of growth performance and the disorder of serum biochemical parameters in DON-challenged piglets. BAI supplementation also alleviated DON-induced negative effects, decreasing protein and gene expression levels of pro-inflammatory cytokines in the serum and intestinal tissue and increasing antioxidant capacity in the serum. BAI increased villus height and villus height/crypt depth but decreased the protein expression levels of nuclear factor kappa B (NF-κB), as determined by immunohistochemical analysis, in the ileum and jejunum. Moreover, we found that BAI inhibited NF-κB and increased mTOR protein and gene expression levels in the serum and intestinal tissues. Collectively, BAI alleviates intestinal inflammatory and oxidative damage by inhibiting NF-κB and increasing mTOR signaling to modulate downstream inflammatory and oxidative responses after DON challenge.

Crucial role of androgen receptor in resistance and endurance trainings-induced muscle hypertrophy through IGF-1/IGF-1R- PI3K/Akt- mTOR pathway

BackgroundAndrogen receptor (AR) has been reported to play vital roles in exercise-induced increase of muscle mass in rats, but needs to be further verified and the mechanism behind remains unclear. As AR target genes, insulin growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) promote muscle hypertrophy through activating PI3K/Akt- mammalian target of rapamycin (mTOR) pathway, a classic pathway of muscle hypertrophy. So the main purpose of this study was using AR antagonist flutamide to demonstrate AR’s effect on training-induced muscle hypertrophy and its possible mechanism: IGF-1/IGF-1R- PI3K/Akt- mTOR pathway?MethodsForty-eight Sprague Dawley male rats aged 7 weeks were randomly divided into six groups: control (C), flutamide (F), resistance training (R), resistance training plus flutamide (R + F), endurance training (E), and endurance training plus flutamide (E + F) groups. Flutamide was used to block AR in rats. Rats in R and R + F groups fulfilled 3 weeks of ladder climbing with progressively increased load, while E and E + F rats completed 3-week moderate intensity aerobic exercise on a treadmill. The relative muscle mass (muscle mass/body weight) of rats was detected. Serum levels of testosterone and IGF-1 of rats were determined by ELISA, and mRNA levels of IGF-1R and mTOR in muscles by real-time PCR. Protein levels of AR, IGF-1, IGF-1R, mTOR, PI3K, Akt, p-PI3K and p-Akt in muscles were detected by Western blot.Results(1) The training-induced rise in the relative muscle mass and the expression levels of AR were only found in the gastrocnemius of R rats and in the soleus of E rats (selective muscle hypertrophy), which were blocked by flutamide. (2) Serum testosterone in the R and E rat were increased, and flutamide exerted no effect. (3) The levels of IGF-1, IGF-1R and mTOR as well as the activities of PI3K and Akt were enhanced selectively (in the gastrocnemius of R rats and in the soleus of E rats), which were reduced by flutamide. Conclusions: AR exerted an essential role in both resistance training and endurance training-induced muscle hypertrophy, which was mediated at least partly through IGF-1/IGF-1R- PI3K/Akt- mTOR pathway.

Bone Marrow-Derived Mesenchymal Stem Cells Combined With Simvastatin Accelerates Burn Wound Healing by Activation of the Akt/mTOR Pathway.

Burn wound healing is one of the most important problems in the field of medical science. Promising results have recently been reported by researchers who used bone marrow mesenchymal stem cells (BMSCs) to treat burn wounds. In this study, we investigated the effects of BMSC therapy in combination with simvastatin (SMV) on angiogenesis as well as on the activity of the Akt/mTOR signaling pathway during burn wound healing in rats. After creating second-degree burn wounds, 40 adult male Wistar rats were randomly divided into four treatment groups: the control, SMV, BMSCs, and the combination therapy group (BMSCs+SMV). Animals were killed 14 days after treatment initiation, and the wounds were removed for histological and molecular analyses. All in all, combination therapy produced better outcomes than individual therapy in terms of the wound closure area, epidermal regeneration level, collagen deposition intensity, and reepithelialization rate. In addition, the elevations of expression levels of Akt and mTOR genes, at both mRNA and protein levels, were more pronounced in the BMSCs+SMV group (P < .05, at least, for both qRT-PCR and western blot assessments). qRT-PCR findings also demonstrated that the wounds treated with the combination of BMSCs and SMV had the highest expression levels of CD31 and VEGF genes (P < .01 for all comparisons). These data suggest that the combined administration of BMSCs transplantation and topical SMV has a great potential in burn wound healing. According to the findings, the beneficial effects of the combination therapy are caused, at least in part, through stimulating Akt/mTOR signaling pathway.

Melatonin regulates the ovarian function and enhances follicle growth in aging laying hens via activating the mammalian target of rapamycin pathway

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.05). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.05). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.

Introduction of Somatic Mutation in MED12 Induces Wnt4/β-Catenin and Disrupts Autophagy in Human Uterine Myometrial Cell.

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/β-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and β-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/β-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages.

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.