Decline in mammary translational capacity during intravenous glucose infusion into lactating dairy cows

The objective of this study was to determine effects of glucose on milk protein yield and mammary mammalian target of rapamycin (mTOR) activity in dairy cattle in early lactation. Eight multiparous cows at 73 ± 8 d in milk were randomly assigned to 2 treatments in a crossover design for two 6-d periods. Treatments were jugular infusion of either saline (Sal) or 896 g/d glucose (Glc). All cows were fed a total mixed ration with 42% neutral detergent fiber, had free access to water, and were milked twice a day. Within each period, blood samples were taken (d 5) and mammary tissue was collected by biopsy (d 6) from each hindquarter for Western blot analysis. In addition to Sal and Glc treatments, on d 6, rapamycin dissolved in 50% dimethyl sulfoxide was administered via the teat canals into the left quarters, with a control solution administered into the right quarters. Rapamycin had no effect on milk protein yields or phosphorylation state of mTOR signaling proteins. Infusions of Glc significantly increased milk yield but only tended to increase milk protein yields. Milk fat tended to be decreased in cows infused with Glc, whereas lactose yields were significantly increased. Glucose infusion did not increase plasma glucose levels, but insulin and nonessential AA concentrations increased by 21 and 16%, respectively, branched-chain AA concentrations decreased 24%, and essential AA concentrations tended to decrease by 14%. Infusion of Glc significantly decreased abundances of both phosphorylated and total ribosomal S6 kinase 1 (S6K1) in mammary tissue by 27 and 11%, respectively. Abundance of phosphorylated eukaryotic initiation factor 4E-binding protein 1 (4EBP1) decreased significantly by 25%, whereas total 4EBP1 exhibited a tendency to decrease by 16%. We conclude that the mTOR signaling pathway is not the only regulator of milk protein synthesis. Decreases in essential AA concentrations in plasma suggest that protein synthesis was stimulated in nonmammary tissues of the body, presumably skeletal muscle.     

New Insights into Red Blood Cell Microcytosis upon mTOR Inhibitor Administration

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 μM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 μM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.     

The Role of AKT/mTOR Pathway in Stress Response to UV-Irradiation: Implication in Skin Carcinogenesis by Regulation of Apoptosis,Autophagy and Senescence

Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis. Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation. Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53. In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway. However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53. In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence. Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival.     

Delicaflavone通过PI3K/AKT/mTOR通路调控上皮-间质转化抑制吉非替尼耐药肺癌PC-9/GR细胞迁移和侵袭

目的：研究Delicaflavone对人吉非替尼耐药肺癌PC-9/GR细胞迁移与侵袭作用及机制。方法：采用MTT法检测Delicaflavone对PC-9/GR细胞存活率的影响;采用细胞划痕实验和Transwell实验测定Delicaflavone对PC-9/GR细胞迁移和侵袭能力的影响;采用Western blotting法检测Delicaflavone对PC-9/GR细胞基质金属蛋白酶9（matrix metalloproteinases 9, MMP-9）、基质金属蛋白酶2（matrix metalloproteinases 2, MMP-2）、钙黏连蛋白（E-cadherin, N-cadherin）、波形蛋白（vimentin）及PI3K/Akt/mTOR信号通路相关蛋白表达情况。结果：与对照组比较,20 mg/L Delicaflavone作用24 h能显著抑制PC-9/GR细胞的细胞活性（P<0.05）,而浓度在10 mg/L以下时则对细胞活性无明显影响。Delicaflavone能够浓度依赖性地抑制PC-9/GR细胞的迁移率、侵袭细胞数量（P<0.05和P<0.01）,并能浓度依赖性地减少PC-9/GR细胞迁移标记蛋白（MMP-9和MMP-2）的表达（P<0.01）,上调E-cadherin、下调N-cadherin和Vimentin的表达（P<0.01）。此外,Delicaflavone还浓度依赖性地减少p-PI3K、p-Akt和p-mTOR表达（P<0.01）。 结论：Delicaflavone具有抑制PC-9/GR细胞迁移及侵袭能力的作用,该作用与Delicaflavone通过PI3K/Akt/mTOR通路调控上皮-间质转化有关。     

Feedback,Crosstalk and Competition: Ingredients for Emergent Non-Linear Behaviour in the PI3K/mTOR Signalling Network

The PI3K/mTOR signalling pathway plays a central role in the governing of cell growth, survival and metabolism. As such, it must integrate and decode information from both external and internal sources to guide efficient decision-making by the cell. To facilitate this, the pathway has evolved an intricate web of complex regulatory mechanisms and elaborate crosstalk with neighbouring signalling pathways, making it a highly non-linear system. Here, we describe the mechanistic biological details that underpin these regulatory mechanisms, covering a multitude of negative and positive feedback loops, feed-forward loops, competing protein interactions, and crosstalk with major signalling pathways. Further, we highlight the non-linear and dynamic network behaviours that arise from these regulations, uncovered through computational and experimental studies. Given the pivotal role of the PI3K/mTOR network in cellular homeostasis and its frequent dysregulation in pathologies including cancer and diabetes, a coherent and systems-level understanding of the complex regulation and consequential dynamic signalling behaviours within this network is imperative for advancing biology and development of new therapeutic approaches.     

Blockade of Serotonin 5-HT6 Receptor Constitutive Activity Alleviates Cognitive Deficits in a Preclinical Model of Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is a common inherited disorder caused by mutations of the NF1 gene that encodes the Ras-GTPase activating protein neurofibromin, leading to overactivation of Ras-dependent signaling pathways such as the mTOR pathway. It is often characterized by a broad range of cognitive symptoms that are currently untreated. The serotonin 5-HT₆ receptor is a potentially relevant target in view of its ability to associate with neurofibromin and to engage the mTOR pathway to compromise cognition in several cognitive impairment paradigms. Here, we show that constitutively active 5-HT₆ receptors contribute to increased mTOR activity in the brain of Nf1^+/− mice, a preclinical model recapitulating some behavioral alterations of NF1. Correspondingly, peripheral administration of SB258585, a 5-HT₆ receptor inverse agonist, or rapamycin, abolished deficits in long-term social and associative memories in Nf1^+/− mice, whereas administration of CPPQ, a neutral antagonist, did not produce cognitive improvement. These results show a key influence of mTOR activation by constitutively active 5-HT₆ receptors in NF1 cognitive symptoms. They provide a proof of concept that 5-HT₆ receptor inverse agonists already in clinical development as symptomatic treatments to reduce cognitive decline in dementia and psychoses, might be repurposed as therapies alleviating cognitive deficits in NF1 patients.     

Haploinsufficiency of a Circadian Clock Gene Bmal1 (Arntl or Mop3) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

Approximately 50–80% of children with autism spectrum disorders (ASDs) exhibit sleep problems, but the contribution of circadian clock dysfunction to the development of ASDs remains largely unknown. The essential clock gene Bmal1 (Arntl or Mop3) has been associated with human sociability, and its missense mutation is found in ASD. Our recent study found that Bmal1-null mice exhibit a variety of autism-like phenotypes. Here, we further investigated whether an incomplete loss of Bmal1 function could cause significant autism-like behavioral changes in mice. Our results demonstrated that heterozygous Bmal1 deletion (Bmal1^+/−) reduced the Bmal1 protein levels by ~50–75%. Reduced Bmal1 expression led to decreased levels of clock proteins, including Per1, Per2, Cry 1, and Clock but increased mTOR activities in the brain. Accordingly, Bmal1^+/− mice exhibited aberrant ultrasonic vocalizations during maternal separation, deficits in sociability and social novelty, excessive repetitive behaviors, impairments in motor coordination, as well as increased anxiety-like behavior. The novel object recognition memory remained intact. Together, these results demonstrate that haploinsufficiency of Bmal1 can cause autism-like behavioral changes in mice, akin to those identified in Bmal1-null mice. This study provides further experimental evidence supporting a potential role for disrupted clock gene expression in the development of ASD.     

Mechanisms of Sensitivity and Resistance of Primary Effusion Lymphoma to Dimethyl Fumarate (DMF)

PEL is a rare B cell lymphoma associated with KSHV that mainly arises in immune-deficient individuals. The search for new drugs to treat this cancer is still ongoing given its aggressiveness and the poor response to chemotherapies. In this study, we found that DMF, a drug known for its anti-inflammatory properties which is registered for the treatment of psoriasis and relapsing–remitting MS, could be a promising therapeutic strategy against PEL. Indeed, although some mechanisms of resistance were induced, DMF activated NRF2, reduced ROS and inhibited the phosphorylation of STAT3 and the release of the pro-inflammatory and immune suppressive cytokines IL-6 and IL-10, which are known to sustain PEL survival. Interestingly, we observed that DMF displayed a stronger cytotoxic effect against fresh PEL cells in comparison to PEL cell lines, due to the activation of ERK1/2 and autophagy in the latter cells. This finding further encourages the possibility of using DMF for the treatment of PEL.     

Characterization of the Basal and mTOR-Dependent Acute Pulmonary and Systemic Immune Response in a Murine Model of Combined Burn and Inhalation Injury

Severe burn injury leads to a cascade of local and systemic immune responses that trigger an extreme state of immune dysfunction, leaving the patient highly susceptible to acute and chronic infection. When combined with inhalation injury, burn patients have higher mortality and a greater chance of developing secondary respiratory complications including infection. No animal model of combined burn and inhalation injury (B+I) exists that accurately mirrors the human clinical picture, nor are there any effective immunotherapies or predictive models of the risk of immune dysfunction. Our earlier work showed that the mechanistic/mammalian target of rapamycin (mTOR) pathway is activated early after burn injury, and its chemical blockade at injury reduced subsequent chronic bacterial susceptibility. It is unclear if mTOR plays a role in the exacerbated immune dysfunction seen after B+I injury. We aimed to: (1) characterize a novel murine model of B+I injury, and (2) investigate the role of mTOR in the immune response after B+I injury. Pulmonary and systemic immune responses to B+I were characterized in the absence or presence of mTOR inhibition at the time of injury. Data describe a murine model of B+I with inhalation-specific immune phenotypes and implicate mTOR in the acute immune dysfunction observed.     

The mTOR Signaling Pathway in Multiple Sclerosis; from Animal Models to Human Data

This article recapitulates the evidence on the role of mammalian targets of rapamycin (mTOR) complex pathways in multiple sclerosis (MS). Key biological processes that intersect with mTOR signaling cascades include autophagy, inflammasome activation, innate (e.g., microglial) and adaptive (B and T cell) immune responses, and axonal and neuronal toxicity/degeneration. There is robust evidence that mTOR inhibitors, such as rapamycin, ameliorate the clinical course of the animal model of MS, experimental autoimmune encephalomyelitis (EAE). New, evolving data unravel mechanisms underlying the therapeutic effect on EAE, which include balance among T-effector and T-regulatory cells, and mTOR effects on myeloid cell function, polarization, and antigen presentation, with relevance to MS pathogenesis. Radiologic and preliminary clinical data from a phase 2 randomized, controlled trial of temsirolimus (a rapamycin analogue) in MS show moderate efficacy, with significant adverse effects. Large clinical trials of indirect mTOR inhibitors (metformin) in MS are lacking; however, a smaller prospective, non-randomized study shows some potentially promising radiological results in combination with ex vivo beneficial effects on immune cells that might warrant further investigation. Importantly, the study of mTOR pathway contributions to autoimmune inflammatory demyelination and multiple sclerosis illustrates the difficulties in the clinical application of animal model results. Nevertheless, it is not inconceivable that targeting metabolism in the future with cell-selective mTOR inhibitors (compared to the broad inhibitors tried to date) could be developed to improve efficacy and reduce side effects.