S6K1 Expression Research Articles

Partial replacement of fishmeal by fermented soybean meal in diets for juvenile white shrimp (Litopenaeus vannamei)

As an important feed ingredient, fermented soybean meal (FSM) has been widely used in aquatic animals due to its stable sources and reasonable price. Here, we evaluated the potential for replacement of fishmeal (FM) with FSM in diet of Litopenaeus vannamei. Five isonitrogenous (410 g/kg) and isolipidic (80 g/kg) diets were formulated: a control diet containing 320 g/kg FM and four experimental diets in which FM in control diet was replaced by FSM at 10 (FSM10%), 20 (FSM20%), 30 (FSM30%) and 40% (FSM40%). An 8-week feeding trial was conducted in fifteen fibreglass tanks with 50 shrimps per tank. After 8 weeks trial, FSM20% had significantly enhanced growth performance (p 0.05). Through real-time quantitative PCR analysis, tor and s6k expression levels of FSM20% were significantly up-regulated (p 0.05), which suggested mTOR signalling pathway was not affected by FSM diets. Based on the above data, 20% replacement of FM with FSM was reasonable and advantageous for L. vannamei diet.

Protein 14-3-3ε Regulates Cell Proliferation and Casein Synthesis via PI3K-mTOR Pathway in Dairy Cow Mammary Epithelial Cells.

Cell proliferation and casein synthesis of dairy cow mammary epithelial cells (DCMECs) are regulated by many factors. This research aimed to investigate the effect of 14-3-3ε on cell proliferation and casein synthesis in DCMECs and to reveal the underlying mechanism. Overexpressing or inhibiting 14-3-3ε demonstrated that cell proliferation; casein synthesis; expression of mTOR, p-mTOR, S6K1, and p-S6K1; and lysosomal localization of mTOR were all up-regulated by 14-3-3ε overexpressing and down-regulated by 14-3-3ε inhibiting. In addition, inhibiting mTOR demonstrated that the up-regulation of cell proliferation and casein synthesis in response to 14-3-3ε overexpressing was removed by inhibiting mTOR. Furthermore, the regulatory mechanism of 14-3-3ε was analyzed by coimmunoprecipitation, and we found that 14-3-3ε could interact with PI3K and activate mTORC1 pathway via PI3K. In addition, DCMECs were treated with insulin and prolactin, and the result showed that the cell proliferation and the expression of CSN2 and 14-3-3ε were all up-regulated by these hormones. In conclusion, the current research showed that 14-3-3ε is an important positive regulatory factor for cell proliferation and casein synthesis in DCMECs, as it up-regulates cell proliferation and casein synthesis via activating PI3K-mTOR pathway.

Growth Performance, Digestive Enzymes, and TOR Signaling Pathway of Litopenaeus vannamei Are Not Significantly Affected by Dietary Protein Hydrolysates in Practical Conditions.

Protein hydrolysates have been widely reported as the protein source of aquatic feed. However, previous studies on protein hydrolysates focused on fish under experimental conditions. In this study, a 6-week feeding trial in a greenhouse was conducted to investigate the effects of partially replacing fishmeal by protein hydrolysates on growth performance, digestive enzymes, and TOR signaling pathway of Litopenaeus vannamei under practical conditions. This involved randomly selecting 72,000 shrimps (initial body weight 2.26 ± 0.02 g) and placing them in groups inside nine concrete tanks (L 5 m × W 5 m × H 1 m) (3 treatments × 3 replicates × 8000 individuals per concrete tank). Two isonitrogenous (430 g kg-1) and isolipidic (80 g kg-1) diets were prepared: fishmeal diet (FM) containing 400g kg-1 fishmeal, and protein hydrolysates diet (PH) in which 15% of the fishmeal was replaced by protein hydrolysates. A commercial diet (CD) was used as reference. The final weight (FW), percent weight gain (PWG), specific growth ratio (SGR), and total weight for each tank (TW) of L. vannamei fed with FM and PH diets were not significantly different (P > 0.05). However, shrimp fed with PH diet had significantly higher FW, PWG, SGR, and TW values than those fed with CD diet (P < 0.05). Trypsin activity was significantly lower in shrimp fed with CD diet, than in shrimp fed with FM and PH diets (P < 0.05). However, trypsin activity of L. vannamei fed with FM and PH diets were not significantly different (P > 0.05). The mRNA expression of tor, s6k, and 4e-bp genes were not significantly affected between FM and PH diets, while tor and s6k expression levels of CD diet were significantly down-regulated. Based on the above data, 15% replacement of fishmeal with protein hydrolysates did not make any difference on shrimps compared with FM group. Therefore, protein hydrolysates can partially replace fishmeal as the protein source of shrimp formula feed in practical conditions.

Comparison of effects of oleic acid and palmitic acid on lipid deposition and mTOR / S6K1 / SREBP-1c pathway in HepG2 cells

Objective: To explore the effects of oleic acid and palmitic acid on lipid deposition and mTOR/S6K1/SREBP-1c pathways in HepG2 cells. Methods: The model of steatosis was established with induction of oleic acid and palmitic acid and was intervened by rapamycin. The changes in lipid droplets were observed after staining the cells with oil Red O. Intracellular triglyceride (TG) contents in cells were measured by TG kit. mTOR, S6K1, and SREBP-1c mRNA expression levels were detected using QRT-PCR. Western blot was used to determine protein expression levels of mTOR, S6K1 and SREBP-1c. Results: Both fatty acids increased lipid droplets in HepG2 cells. Fatty degeneration with elevated TG occurred with significant changes in oleic acid group lipids. Rapamycin alleviated lipid deposition caused by oleic acid and palmitic acid and inhibited their induction of increased expression of mTOR, S6K1, and SREBP-1c. QRT-PCR and Western blot results showed that mRNA and protein expressions of mTOR, S6K1, and SREBP-1c in oleic acid and palmitic acid group were significantly higher than the control group (P < 0.05). The increase was more pronounced in the palmitic acid group (P < 0.05); however, after rapamycin intervention, the expression of mRNA and protein in the three groups were significantly lower (P < 0.05), and the change in palmitic acid group was more pronounced (P < 0.05). Conclusion: Oleic acid and palmitic acid can induce lipid deposition in HepG2 cells and increase expression of every component of mTOR/S6K1/SREBP-1c pathway; however, Oleic acid-induced lipid deposition is more pronounced, and the mTOR, S6K1, and SREBP-1c pathway change is more obvious in palmitic acid. Rapamycin has high potent inhibitory effect on palmitic acid-induced lipid deposition. These results specify that lipid synthesis involved in the mTOR/S6K1/SREBP-1c pathways are mainly associated to palmitic acid in HepG2 cells, whereas other signaling pathway may mediate oleic acid-induced lipid synthesis.

Expression of miRNAs Targeting mTOR and S6K1 Genes of mTOR Signaling Pathway Including miR-96, miR-557, and miR-3182 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer. Aberrant expression of genes in mTOR pathway and their targeting miRNAs plays an important role in TNBC. The aim of this study was to determine the expression of mTOR and S6K1 and their targeting miRNAs in breast cancer cell lines and clinical samples. miRNAs targeting 3'-UTR of mTOR and S6K1 mRNAs were predicted using bioinformatic algorithms. MDA-MB-231, MCF-7, and MCF-10A as well as 20 TNBC samples were analyzed for gene and miRNA expression using quantitative real-time PCR (RT-qPCR). A receiver operating characteristic (ROC) curve analysis was performed for evaluation of candidate miRNAs as diagnostic biomarkers. miR-96 and miR-557 targeting mTOR and S6K1 mRNAs, respectively, were selected, and miR-3182 was selected as the miRNA targeting both genes. The miRNAs were down-regulated in cell lines, while their target mRNAs were up-regulated. Similar findings were observed in clinical samples. The ROC curve analysis revealed decline in expression of these miRNAs. We suggest that miR-96, miR-557, and miR-3182 can be used as inhibitory agents for mTOR and S6K1 in TNBC-targeted therapy.

LXR\u0251 participates in the mTOR/S6K1/SREBP-1c signaling pathway during sodium palmitate-induced lipogenesis in HepG2 cells

BackgroundThe aim of this study was to investigate how the signaling pathway downstream of mTOR/S6K1 contributes to the regulation of SREBP-1c expression during lipogenesis in HepG2 cells.MethodsThe model of steatosis was established using human hepatocytes HepG2 and inducting them with sodium palmitate. mTOR, S6K1 and LXRα were inhibited by rapamycin, PF-4708671 and siRNA-LXRα, respectively. After a variety of different treatment, the levels of intracellular triglycerides, the accumulation of lipid droplets and the expression levels of related genes were detected.ResultsRapamycin, PF-4708671 and siRNA-LXRα treatment could decrease the accumulation of triglycerides and lipid droplets induced by sodium palmitate in HepG2 cells, and the inhibitory effect could be enhanced by the combination of them. Sodium palmitate stimulated the expression of genes encoding mTOR, S6K1, LXRα, SREBP-1c and SREBP-1c target enzymes (FAS and ACC1) in HepG2 cells. Moreover, these genes were sensitive to rapamycin. PF-4708671 also decreased the expression of these genes, except for the mTOR gene, and the extent of reduction could be enhanced by combination with rapamycin. Knockdown of LXRα decreased the expression of SREBP-1c, FAS and ACC1, but it had no effect on the expression of mTOR or S6K1. Furthermore, rapamycin and PF-4708671 enhanced the inhibitory effect of siRNA-LXRα.ConclusionsmTOR/S6K1 regulates the SREBP-1c signaling pathway through LXRα in sodium palmitate-induced HepG2 cells, suggesting LXRα might be a potential therapeutic target for NAFLD.

Insight on ALPPS – Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy – mechanisms: activation of mTOR pathway

BackgroundALPPS procedure has been introduced to increase the volume of future liver remnant. The mechanisms underlying the accelerated regeneration observed with ALPPS are unknown. It was hypothesized that AMPK/mTOR is activated as an integrating pathway for metabolic signals leading to proliferation and cell growth. Our aim was to analyze increase in liver volume, proliferation parameters and expression of AMPK/mTOR pathway-related molecules in patients undergoing ALPPS. MethodsA single center prospective study of patients undergoing ALPPS was performed from 2013 to 2015. Liver and serum samples, clinical laboratory results and CT-scan data were obtained. ELISA, Ki-67 immunostaining and qRT-PCR were performed in deportalized and remnant liver tissue in both stages of the procedure. Results11 patients were enrolled. Remnant liver volume increased 112 ± 63% (p < 0.05) in 9.1 ± 1.6 days. Proliferation-related cytokines IL-6, TNF-α, HGF and EGF significantly increased, while higher Ki-67 immunostaining and cyclin D expression were observed in remnant livers after ALPPS. mTOR, S6K1, 4E-BP1, TSC1 and TSC2 expression were significantly increased in remnant livers at second stage, while AMPK and Akt increased only in deportalized liver samples. ConclusionRapid liver regeneration with ALPPS might be associated with hepatocyte proliferation induced by mTOR pathway activation.

Altered Expression of miR-181a-5p and miR-23a-3p Is Associated With Obesity and TNFα-Induced Insulin Resistance.

The proinflammatory cytokine TNFα is a key player in insulin resistance (IR). The role of miRNAs in inflammation associated with IR is poorly understood. To investigate miR-181a-5p and miR-23a-3p expression profiles in obesity and to study their role in TNFα-induced IR in adipocytes. Two separate cohorts were used. Cohort 1 was used in adipose tissue (AT) expression studies and included 28 subjects with body mass index (BMI) <30 kg/m2 and 30 with BMI ≥30 kg/m2. Cohort 2 was used in circulating serum miRNA studies and included 101 subjects with 4 years of follow-up (48 case subjects and 53 control subjects). miR-181a-5p and miR-23a-3p expression was assessed in subcutaneous and visceral AT. Functional analysis was performed in adipocytes, using miRNA mimics and inhibitors. Key molecules of the insulin pathway, AKT, PTEN, AS160, and S6K, were analyzed. Expression of miR-181a-5p and miR-23a-3p was reduced in adipose tissue from obese and diabetic subjects and was inversely correlated to adiposity and homeostasis model assessment of IR index. Overexpression of miR-181a-5p and miR-23a-3p in adipocytes upregulated insulin-stimulated AKT activation and reduced TNFα-induced IR, regulating PTEN and S6K expression. Serum levels of miR-181a-5p were reduced in case vs control subjects at baseline, suggesting a prognostic value. Variable importance in projection scores revealed miR-181a-5p had more effect on the model than insulin or glucose at 120 minutes. miR-181a-5p and miR-23a-3p may prevent TNFα-induced IR in adipocytes through modulation of PTEN and S6K expression.

Myelodysplastic syndrome among elderly patients with anemia: A Single institutional experience

BACKGROUND AND OBJECTIVES: Many geriatric patients with different cytopenias are symptomatically treated and miss the diagnosis of myelodysplastic syndrome (MDS). The study was done to estimate the prevalence of elderly presenting with cytopenias in hospital population and to assess karyotyping profile and gene expression in mTOR pathway and correlate with clinical course. PATIENTS AND METHODS: Blood samples of patients (> 60 years) who visited outpatient department and who were in - patients being evaluated for anemias, were included. 15 patients were diagnosed with myelodysplastic syndrome. Karyotype profile and gene expression studies for S6K1 and 4E-BP1 and IPSS –R scoring were done. RESULTS: Among 521 patients, normocytic normochromic anemias was most common (48.3%).15 MDS patients were classified (WHO classification) as - Single lineage dysplasia -6, multilineage dysplasia-5 multilineage dysplasia with excess blasts 1- and three with hypo plastic marrow. There was no significant correlation between the karyotype and subgroups of MDS and age. IPSS scoring showed patients, with very low score – n-1 (ARR-1.3) low score n-6 (ARR- 1.6-2.8) intermediate score n-4 (ARR 3.1-3.7). During follow up for two years one death was reported in the intermediate category. Quantification of mRNA expression of S6K1 and 4EBP1 showed absence or reduction in all cases. CONCLUSIONS: An incidence of 2.8% MDS was observed. The median age reported is consistent with Asian literature. IPSS-R scoring with inclusion of cytogenetic analysis helped in effective risk stratification of patients for management. Though no definite conclusions can be drawn on expression of S6K1 and 4EBP1 of mTOR pathway due to limited sample size, their role in the cytopenia cannot be ruled out.

S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer.

Rationale: Mxi1 is regarded as a potential tumor suppressor protein that antagonizes the transcriptional activity of proto-oncogene Myc. However, the clinical significances and underlying mechanisms by which Mxi1 is regulated in lung cancer remain poorly understood.Methods: Mass spectrometry analysis and immunoprecipitation assay were utilized to detect the protein-protein interaction. The phosphorylation of Mxi1 was evaluated by in vitro kinase assays. Poly-ubiquitination of Mxi1 was examined by in vivo ubiquitination assay. Lung cancer cells stably expressing wild-type Mxi1 or Mxi1-S160A were used for functional analyses. The expression levels of Mxi1 and S6K1 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues.Results: We found that Mxi1 is downregulated and correlated with poor prognosis in lung cancer. Using tandem affinity purification technology, we provided evidence that β-Trcp E3 ubiquitin ligase interacts with and promotes the ubiquitination and degradation of Mxi1. Furthermore, we demonstrated that Mxi1 is phosphorylated at S160 site by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase β-Trcp. Moreover, a phosphorylation mutant form of Mxi1 (Mxi1-S160A), which cannot be degraded by S6K1 and β-Trcp, is much more stable and efficient in suppressing the transcriptional activity of Myc and radioresistance in lung cancer cells. More importantly, a strong inverse correlation between S6K1 and Mxi1 expression was observed in human lung cancer tissues.Conclusion: Our findings not only establish a crosstalk between the mTOR/S6K1 signaling pathway and Myc activation, but also suggest that targeting S6K1/Mxi1 pathway is a promising therapeutic strategy for the treatment of lung cancer.

ERR\u03b1 regulates the growth of triple-negative breast cancer cells via S6K1-dependent mechanism

Estrogen-related receptor alpha (ERRα) is an orphan nuclear factor that is a master regulator of cellular energy metabolism. ERRα is overexpressed in a variety of tumors, including ovarian, prostate, colorectal, cervical and breast, and is associated with a more aggressive tumor and a worse outcome. In breast cancer, specifically, high ERRα expression is associated with an increased rate of recurrence and a poor prognosis. Because of the common functions of ERRα and the mTORC1/S6K1 signaling pathway in regulation of cellular metabolism and breast cancer pathogenesis, we focused on investigating the biochemical relationship between ERRα and S6K1. We found that ERRα negatively regulates S6K1 expression by directly binding to its promoter. Downregulation of ERRα expression sensitized ERα-negative breast cancer cells to mTORC1/S6K1 inhibitors. Therefore, our results show that combinatorial inhibition of ERRα and mTORC1/S6K1 may have clinical utility in treatment of triple-negative breast cancer, and warrants further investigation.

FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells

Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26–34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system.

Metformin inhibited esophageal squamous cell carcinoma proliferation in vitro and in vivo and enhanced the anti-cancer effect of cisplatin.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor prognosis in China. Chemotherapy now is one of the most frequently used treatments for patients with ESCC in middle or late stage, however the effects were often limited by increased chemoresistance or treatment toxicity. So it is urgent to find new drugs to treat ESCC patients. Metformin with low cost and toxicity has proved to have anti-cancer effects in a numerous cancers, while its role and mechanism in ESCC has seldom been studied. In the present study, we found that metformin exhibited not only an anti-proliferation ability in a dose and time dependent manner but also a proapoptosis effect in a dose dependent manner in ESCC cell line KYSE450. Our in vivo experiment also showed that metformin markedly inhibited KYSE450 xenograft tumors growth compared to those treated with normal saline. What’s more, no obvious toxic reactions were observed. To further explore the underlying mechanism, we found that metformin treatment could significantly damp the expression of 4EBP1 and S6K1 in KYSE 450 cells in vitro and in vivo, furthermore, the p-4EBP1 and p-S6K1 expression in KYSE 450 cells were also inhibited greatly in vitro and in vivo. During the therapy of cancer, in order to overcome side effects, combination therapy was often used. In this paper, we demonstrated that metformin potentiated the effects of cisplatin via inhibiting cell proliferation and promoting cell apoptosis. Taken together, metformin owned the potential anti-cancer effect on ESCC in monotherapy or was combined with cisplatin and these results laid solid basis for the use of metformin in ESCC.

Alterations of mTOR pathway and autophagy in early type 2 diabetic cardiomyopathy in rats

Objective: To investigate the alterations of mTOR signaling pathway and autophagy in the development of type 2 diabetes and early diabetic cardiomyopathy and to study their roles in pathogenesis of diabetic myocardium. Methods: A type 2 diabetes rat model was established by injection of streptozocin after five-week of high fat diet. The rats were randomly divided into control group, experiment group of 2 weeks and experiment group of 4 weeks. Alterations of mTOR, p-mTOR, S6K1, Beclin-1 and LC3-Ⅱ expression in myocardium were determined by Western blot and immunohistochemistry. Results: Compared with the control group, the expression of mTOR, p-mTOR, S6K1, Beclin-1 and LC3-Ⅱ level increased significantly in the experiment group of 2 weeks. The expression of p-mTOR and S6K1 increased significantly in the experiment group of 4 weeks compared with those of the experiment group of 2 weeks. Conclusions: mTOR signaling pathway is activated in early diabetic myocardial injury via autophagy. The findings may provide a new therapeutic target for diabetic cardiomyopathy.

Lactobacilli Differentially Modulate mTOR and Wnt/ β-Catenin Pathways in Different Cancer Cell Lines.

BackgroundLactobacilli are a group of beneficial bacteria whose anti cancer effects have been evaluated in different cancer cell lines as well as animal models and human subjects. Such anti cancer effects can be exerted via different mechanisms such as modulation of immune response as well as inhibition of pathogens colonization. In addition, lactobacilli have direct cytotoxic effects against cancer cells which may be exerted through modulation of expression cancer related pathways.ObjectivesThe aim of this study is to find the mechanism of anti cancer effects of two lactobacilli strains, Lactobacillus. crispatus (LC) and Lactobacillus. rhamnosus (LR).Materials and MethodsWe analyzed expression of some mTOR and Wnt/ β-catenin pathways genes in three cancer cell lines (HeLa, MDA-MB-231 and HT-29) following treatment with LC and LR culture supernatants.ResultsOf note, the expression of CCND1 as a marker of cell proliferation, survival, and angiogenesis, has been decreased following LR treatment in all cell lines. In addition, the expression of SFRP2, an antagonist of Wnt pathway, has been increased in HT-29 following LR treatment and in HeLa cells following LR and LC treatments. Furthermore, we have demonstrated the downregulation of S6K1 expression, a marker of poor prognosis, following LR treatment in HT-29 and following LR and LC treatments in MDA-MB-231 cell line.ConclusionsConsequently, lactobacilli can modulate expression of mTOR and Wnt/ β-catenin pathways genes in cancer cell lines in a strain specific as well as cell type specific manner.

Impact of dietary branched chain amino acids concentration on broiler chicks during aflatoxicosis

A 20-day trial was conducted to determine the effects of dietary branched-chain amino acids (BCAA) on performance, nutrient digestibility, and gene expression of the mTOR pathway in broiler chicks when exposed to aflatoxin B1 (AFB1). The 6 dietary treatments were arranged in a 2 × 3 factorial with 3 BCAA concentrations (1.16, 1.94, and 2.73%) with or without 1.5 mg/kg AFB1 (1.77 mg/kg analyzed). Each diet was fed to 8 replicate cages (6 chicks per cage) from 6 to 20 d of age. Exposure to AFB1 significantly reduced gain:feed ratio and breast muscle weight (P < 0.05), and tended to decrease cumulative BW gain (P = 0.087), while increasing dietary BCAA improved all performance measures (P ≤ 0.0002), except relative breast muscle weight. Apparent ileal digestibility of N and 9 amino acids were increased by AFB1 (P ≤ 0.05), but were reduced by higher dietary BCAA (P ≤ 0.023). Jejunum histology was not affected by AFB1, while higher dietary BCAA tended to increase villus height (P = 0.08). Additionally, the gene expression of mTOR pathway (mTOR, 4EBP1, and S6K1) from liver and jejunum were not affected by dietary treatments, while muscle expression of S6K1 tended to be increased by AFB1 (P = 0.07). No significant interaction between AFB1 and dietary BCAA were observed for any measures in the current study. Results from this study suggested that feed AFB1 contamination can significantly reduce growth performance and breast muscle growth in broiler chicks at 20 d. Higher BCAA supply may have beneficial impact on bird performance, but this effect is independent of AFB1 exposure.

Zinc stimulates glucose consumption by modulating the insulin signaling pathway in L6 myotubes: essential roles of Akt–GLUT4, GSK3β and mTOR–S6K1

The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3β), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3β phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3β phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt–GLUT4, GSK3β, mTOR and S6K1.

Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR.

As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

Effects of treadmill exercise on skeletal muscle mTOR signaling pathway in high-fat diet-induced obese mice.

[Purpose] The aim of this study was to investigate the effects of regular treadmill exercise on skeletal muscle Rictor-Akt and mTOR-Raptor-S6K1 signaling pathway in high-fat diet-induced obese mice. [Subjects and Methods] Four- week-old C57BL/6 mice were adopted and classified into normal diet group (ND, n = 10), normal diet and training group (NDT, n = 10), high-fat diet group (HF, n = 10), and high-fat diet and training group (HFT, n = 10). The exercise program consisted of a treadmill exercise provided at low intensity for 1–4 weeks, and moderate intensity for 5–8 weeks. [Results] The Western blot method was used to measure the expression of mTOR, Raptor, S6K1, Rictor, and Akt proteins in the soleus muscle. mTOR levels were significantly higher in the HF group than in the ND and NDT groups. Raptor/mTORC1 and S6K1 levels were significantly higher in the HF group than in all the other groups. Akt levels were significantly lower in the HF group than in the NDT group. The risk of obesity may be associated with the overactivation of the mTOR-Raptor-S6K1 signaling pathway and a decrease in Akt levels. [Conclusion] This study also indicates that performing aerobic exercise may be associated with the downregulation of the mTOR-Raptor-S6K1 pathway.

Effects of dietary leucine supplementation on the gene expression of mammalian target of rapamycin signaling pathway and intestinal development of broilers.

This experiment was to investigate the effects of dietary leucine supplementation on the gene expression of mammalian target of rapamycin (mTOR) signaling pathway and intestinal development of broilers. A total of 384 one-day-old broilers were randomly assigned into 4 treatments with 6 replicates (16 broilers per replicate). Broilers in these treatment groups were offered the following diets with 1.37, 1.77, 2.17 and 2.57% of leucine. These diet treatments were named 1.37TM, 1.77TM, 2.17TM, and 2.57TM. The experiment lasted 21 days and all birds had free access to feed and water. Results indicated that there was no significant difference in body weight, average daily gain and average feed intake among all treatments (P > 0.05). The broiler duodenal villus height in 2.57TM was the lowest, but the highest occurred in 1.37TM on d 7 and 14 (P < 0.05). The villus height in the jejunum and ileum increased along with leucine level from 1.37 to 2.17%. The villus height of jejunum was significantly higher in 2.17TM than in 1.37TM on d 7 and 14, and the ratio of villus height to crypt depth (V:C) in the duodenum, jejunum and ileum increased significantly (P < 0.05) on d 21. The gene expression level of mTOR in the duodenum decreased with increasing leucine level and was higher in 1.37TM than in 2.57TM on d 7 and 14 (P < 0.05). On d 14 and 21 of the trial, the expression of S6K1 in the duodenum was higher in 1.37TM than in 2.57TM (P < 0.05), and the expression of mTOR, S6K1 in the jejunum and ileum increased with increasing leucine level form 1.37 to 2.17%, whereas a significant difference occurred between 1.37TM and 2.17TM (P < 0.05). In conclusion, the addition of leucine fails to enhance the growth performance of broilers. However, leucine can improve intestinal development by enhancing villus height and V:C ratio in the jejunum and ileum. Moreover, the expression of mTOR, S6K1 increased as the level of dietary leucine was elevated from 1.37 to 2.17%.