The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.     

Mouse embryos used as a bioassay to determine control of marsupial embryonic diapause

Mouse blastocysts appear to be under direct inhibition from the uterine environment, whereas no evidence of direct inhibition during diapause in the tammar wallaby has been observed. Normally developing (day 4) and quiescent mouse blastocysts were incubated for up to 12 hr in media supplemented with BSA, wallaby plasma, wallaby day 0 (day of removal of pouch young; RPY), day 5, or day 10 endometrial exudates at a concentration of 2 mg/ml of protein, and analyzed for rates of carbohydrate metabolism using fluorescence and radioisotopes. Rates of glucose uptake and lactate production by day 4 blastocysts increase after incubation with day 10 and day 5 wallaby exudates compared with rates by blastocysts incubated in BSA. Pyruvate uptake increased after 8 hr irrespective of incubation media, except for embryos incubated in day 0 exudate, which maintained levels significantly lower than BSA-incubated embryos. Quiescent mouse embryos displayed a high ATP/ADP ratio during diapause (1.06 +/- 0.24) which decreased after 4 hr incubation in all media (0.42 +/- 0.05; P < 0.01) but embryos incubated in day 0 exudate media remained at a significantly higher level than embryos incubated in BSA. These results indicate that quiescent tammar endometrial exudate is not capable of initiating diapause in mouse embryos at the concentration used, but is able to slow the rate of reactivation of quiescent blastocysts. Importantly, reactivated wallaby exudate increases mouse blastocyst glucose metabolism and lactate production. It is possible that the quiescent tammar endometrial environment has an inhibitory factor necessary to maintain diapause in the tammar blastocyst.     

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient‐sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB‐4, HL‐60 and KG‐1) and their impact on autophagy and survival was characterized. Data show that whereas KG‐1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co‐activation of AMPK and mTORC1 associated with increased autophagy, NB‐4 and HL‐60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG‐1 cells’ survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti‐leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.     

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear.In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary – and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies.All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation.It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.     

Chronic hypoxia-induced alterations of key enzymes of glucose oxidative metabolism in developing mouse liver are mTOR dependent

Hypoxia is a potent regulator of gene expression and cellular energy metabolism and known to interfere with post-natal growth and development. Although hypoxia can induce adaptive changes in the developing liver, the mechanisms underlying these changes are poorly understood. To elucidate some of the adaptive changes chronic hypoxia induces in the developing liver, we studied the expression of the genes of mammalian target of rapamycin (mTOR) signaling and glucose metabolism, undertook proteomic examination with 2D gel-MS/MS of electron transport chain, and determined activities and protein expression of several key regulatory enzymes of glucose oxidative metabolism. To gain insight into the molecular mechanism underlying hypoxia-induced liver metabolic adaptation, we treated a subset of mice with rapamycin (0.5 mg/kg/day) to inhibit mTOR postnatally. Rapamycin-treated mice showed lower birth weight, lower body weight, and liver growth retardation in a pattern similar to that observed in the hypoxic mice at P30. Rapamycin treatment led to differential impact on the cytoplasmic and mitochondrial pathways of glucose metabolism. Our results suggest a decrease in mTOR activity as part of the mechanisms underlying hypoxia-induced changes in the activities of glycolytic and TCA cycle enzymes in liver. Chronic postnatal hypoxia induces mTOR-dependent differential effects on liver glycolytic and TCA cycle enzymes and as such should be studied further as they have pathophysiological implications in hepatic diseases and conditions in which hypoxia plays a role.     

Metabolic rewiring in cancer cells overexpressing the glucocorticoid-induced leucine zipper protein (GILZ): Activation of mitochondrial oxidative phosphorylation and sensitization to oxidative cell death induced by mitochondrial targeted drugs

Cancer cell metabolism is largely controlled by oncogenic signals and nutrient availability. Here, we highlighted that the glucocorticoid-induced leucine zipper (GILZ), an intracellular protein influencing many signaling pathways, reprograms cancer cell metabolism to promote proliferation. We provided evidence that GILZ overexpression induced a significant increase of mitochondrial oxidative phosphorylation as evidenced by the augmentation in basal respiration, ATP-linked respiration as well as respiratory capacity. Pharmacological inhibition of glucose, glutamine and fatty acid oxidation reduced the activation of GILZ-induced mitochondrial oxidative phosphorylation. At glycolysis level, GILZ-overexpressing cells enhanced the expression of glucose transporters in their plasmatic membrane and showed higher glycolytic reserve. ¹H NMR metabolites quantification showed an up-regulation of amino acid biosynthesis. The GILZ-induced metabolic reprograming is present in various cancer cell lines regardless of their driver mutations status and is associated with higher proliferation rates persisting under metabolic stress conditions. Interestingly, high levels of OXPHOS made GILZ-overexpressing cells vulnerable to cell death induced by mitochondrial pro-oxidants. Altogether, these data indicate that GILZ reprograms cancer metabolism towards mitochondrial OXPHOS and sensitizes cancer cells to mitochondria-targeted drugs with pro-oxidant activities.     

Neural stem/progenitor cells display a low requirement for oxidative metabolism independent of hypoxia inducible factor‐1alpha expression

Neural stem/progenitor cells (NSPCs) are multipotent cells within the embryonic and adult brain that give rise to both neuronal and glial cell lineages. Maintenance of NSPC multipotency is promoted by low oxygen tension, although the metabolic underpinnings of this trait have not been described. In this study, we investigated the metabolic state of undifferentiated NSPCs in culture, and tested their relative reliance on oxidative versus glycolytic metabolism for survival, as well as their dependence on hypoxia inducible factor‐1alpha (HIF‐1α) expression for maintenance of metabolic phenotype. Unlike primary neurons, NSPCs from embryonic and adult mice survived prolonged hypoxia in culture. In addition, NSPCs displayed greater susceptibility to glycolytic inhibition compared with primary neurons, even in the presence of alternative mitochondrial TCA substrates. NSPCs were also more resistant than neurons to mitochondrial cyanide toxicity, less capable of utilizing galactose as an alternative substrate to glucose, and more susceptible to pharmacological inhibition of the pentose phosphate pathway by 6‐aminonicotinamide. Inducible deletion of exon 1 of the Hif1a gene improved the ability of NSPCs to utilize pyruvate during glycolytic inhibition, but did not alter other parameters of metabolism, including their ability to withstand prolonged hypoxia. Taken together, these data indicate that NSPCs have a relatively low requirement for oxidative metabolism for their survival and that hypoxic resistance is not dependent upon HIF‐1α signaling.     

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10⁰ TCID₅₀ MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10⁻⁵ TCID₅₀ MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.     

Metabolic Reprogramming towards Aerobic Glycolysis Correlates with Greater Proliferative Ability and Resistance to Metabolic Inhibition in CD8 versus CD4 T Cells

T lymphocytes (T cells) undergo metabolic reprogramming after activation to provide energy and biosynthetic materials for growth, proliferation and differentiation. Distinct T cell subsets, however, adopt metabolic programs specific to support their needs. As CD4 T cells coordinate adaptive immune responses while CD8 T cells become cytotoxic effectors, we compared activation-induced proliferation and metabolic reprogramming of these subsets. Resting CD4 and CD8 T cells were metabolically similar and used a predominantly oxidative metabolism. Following activation CD8 T cells proliferated more rapidly. Stimulation led both CD4 and CD8 T cells to sharply increase glucose metabolism and adopt aerobic glycolysis as a primary metabolic program. Activated CD4 T cells, however, remained more oxidative and had greater maximal respiratory capacity than activated CD8 T cells. CD4 T cells were also associated with greater levels of ROS and increased mitochondrial content, irrespective of the activation context. CD8 cells were better able, however, to oxidize glutamine as an alternative fuel source. The more glycolytic metabolism of activated CD8 T cells correlated with increased capacity for growth and proliferation, along with reduced sensitivity of cell growth to metabolic inhibition. These specific metabolic programs may promote greater growth and proliferation of CD8 T cells and enhance survival in diverse nutrient conditions.     

Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling.     

Complex I dysfunction redirects cellular and mitochondrial metabolism in Arabidopsis

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. However, the plant enzyme has structural and functional features that set it apart from its counterparts in other organisms, raising questions about the physiological significance of this complex in plants. We have developed an experimental model in which rotenone, a classic complex I inhibitor, has been applied to Arabidopsis (Arabidopsis thaliana) cell suspension cultures in order to dissect early metabolic adjustments involved in cell acclimation to mitochondrial dysfunction. Rotenone induced a transitory decrease in cellular respiration (0-4 h after treatment). Cell respiration then progressively recovered and reached a steady state at 10 to 12 h after treatment. Complex I inhibition by rotenone did not induce obvious oxidative stress or cell death but affected longer term cell growth. Integrated analyses of gene expression, the mitochondrial proteome, and changes in primary metabolism indicated that rotenone treatment caused changes in mitochondrial function via alterations in specific components. A physical disengagement of glycolytic activities associated with the mitochondrial outer membrane was observed, and the tricarboxylic acid cycle was altered. Amino acid and organic acid pools were also modified by rotenone treatment, with a marked early decrease of 2-oxoglutarate, aspartate, and glutamine pools. These data demonstrate that, in Arabidopsis cells, complex I inhibition by rotenone induces significant remodeling of metabolic pathways involving the mitochondria and other compartments and point to early metabolic changes in response to mitochondrial dysfunction.     

Energy metabolism in tumor cells

In early studies on energy metabolism of tumor cells, it was proposed that the enhanced glycolysis was induced by a decreased oxidative phosphorylation. Since then it has been indiscriminately applied to all types of tumor cells that the ATP supply is mainly or only provided by glycolysis, without an appropriate experimental evaluation. In this review, the different genetic and biochemical mechanisms by which tumor cells achieve an enhanced glycolytic flux are analyzed. Furthermore, the proposed mechanisms that arguably lead to a decreased oxidative phosphorylation in tumor cells are discussed. As the O(2) concentration in hypoxic regions of tumors seems not to be limiting for the functioning of oxidative phosphorylation, this pathway is re-evaluated regarding oxidizable substrate utilization and its contribution to ATP supply versus glycolysis. In the tumor cell lines where the oxidative metabolism prevails over the glycolytic metabolism for ATP supply, the flux control distribution of both pathways is described. The effect of glycolytic and mitochondrial drugs on tumor energy metabolism and cellular proliferation is described and discussed. Similarly, the energy metabolic changes associated with inherent and acquired resistance to radiotherapy and chemotherapy of tumor cells, and those determined by positron emission tomography, are revised. It is proposed that energy metabolism may be an alternative therapeutic target for both hypoxic (glycolytic) and oxidative tumors.     

Mammalian target of rapamycin promotes vincristine resistance through multiple mechanisms independent of maintained glycolytic rate

Deregulation of the phosphoinositide 3-kinase-Akt pathway is a major contributor to oncogenesis and resistance to cancer therapy. Recent work has shown mammalian target of rapamycin (mTOR) to be a major target downstream of Akt that contributes to both transformation and therapeutic resistance. Although inhibitors of Akt are not yet clinically available, rapamycin, a mTOR-specific inhibitor, has long been used as an immunosuppressant, and several rapamycin analogues are now in clinical trials in oncology. Recent data indicate that a mTOR complex phosphorylates Akt, and this complex is insensitive to rapamycin. We show that dominant-negative mTOR diminishes phosphorylation of endogenous Akt and exogenous myristoylated Akt (mAkt), that prolonged exposure to rapamycin also inhibits Akt activation, and that this inhibition is dependent on new protein synthesis. These data suggest that mTOR facilitates Akt activation through mechanisms other than direct phosphorylation. A constitutively active mTOR mutant that fails to enhance Akt phosphorylation nevertheless promotes resistance to multiple antimicrotubule agents, indicating that mTOR also mediates survival independent of Akt. Although Akt- and mTOR-mediated survival has been linked to regulation of cellular metabolism, we also show that survival and metabolic control are separable. The hexokinase inhibitor 5-thioglucose markedly inhibits glycolytic rate but does not diminish vincristine resistance mediated by mAkt or mTOR, and it has only a minor effect on mTOR- or mAkt-mediated resistance to growth factor withdrawal, suggesting that Akt-mTOR-mediated resistance is largely independent of maintenance of glycolytic rate. We conclude that mTOR activity can promote resistance through multiple mechanisms independent of maintained glycolytic rate.     

Suppressed glycolytic metabolism in the prostate of transgenic rats overexpressing calcium-binding protein regucalcin underpins reduced cell proliferation

Regucalcin (RGN) is a calcium-binding protein underexpressed in human prostate cancer cases, and it has been associated with the suppression of cell proliferation and the regulation of several metabolic pathways. On the other hand, it is known that the metabolic reprogramming with augmented glycolytic metabolism and enhanced proliferative capability is a characteristic of prostate cancer cells. The present study investigated the influence of RGN on the glycolytic metabolism of rat prostate by comparing transgenic adult animals overexpressing RGN (Tg-RGN) with their wild-type counterparts. Glucose consumption was significantly decreased in the prostate of Tg-RGN animals relatively to wild-type, and accompanied by the diminished expression of glucose transporter 3 and glycolytic enzyme phosphofructokinase. Also, prostates of Tg-RGN animals displayed lower lactate levels, which resulted from the diminished expression/activity of lactate dehydrogenase. The expression of the monocarboxylate transporter 4 responsible for the export of lactate to the extracellular space was also diminished with RGN overexpression. These results showed the effect of RGN in inhibiting the glycolytic metabolism in rat prostate, which was underpinned by a reduced cell proliferation index. The present findings also suggest that the loss of RGN may predispose to a hyper glycolytic profile and fostered proliferation of prostate cells.     

Embryonic diapause is conserved across mammals

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.     

Antagonistic crosstalk between NF-κB and SIRT1 in the regulation of inflammation and metabolic disorders

Recent studies have indicated that the regulation of innate immunity and energy metabolism are connected together through an antagonistic crosstalk between NF-κB and SIRT1 signaling pathways. NF-κB signaling has a major role in innate immunity defense while SIRT1 regulates the oxidative respiration and cellular survival. However, NF-κB signaling can stimulate glycolytic energy flux during acute inflammation, whereas SIRT1 activation inhibits NF-κB signaling and enhances oxidative metabolism and the resolution of inflammation. SIRT1 inhibits NF-κB signaling directly by deacetylating the p65 subunit of NF-κB complex. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PGC-1α and simultaneously, these factors inhibit NF-κB signaling and suppress inflammation. On the other hand, NF-κB signaling down-regulates SIRT1 activity through the expression of miR-34a, IFNγ, and reactive oxygen species. The inhibition of SIRT1 disrupts oxidative energy metabolism and stimulates the NF-κB-induced inflammatory responses present in many chronic metabolic and age-related diseases. We will examine the molecular mechanisms of the antagonistic signaling between NF-κB and SIRT1 and describe how this crosstalk controls inflammatory process and energy metabolism. In addition, we will discuss how disturbances in this signaling crosstalk induce the appearance of chronic inflammation in metabolic diseases.