Shift In Cellular Metabolism Research Articles

The Metabolic Phenotype Associated with Mounting an Immune Response to a Systemic Infection of Listeria Monocytogenes (FS12-07-19)

ObjectivesOur goal in these studies was to quantitatively determine the metabolic phenotype of intracellular bacterial infection, immune response, and clearance. Mounting an immune response to a bacterial infection is metabolically taxing to the host. During infection, the host exhibits sickness syndrome characterized by fever, lethargy and anorexia. Cells of the immune system also shift cellular metabolic pathways, which alters the metabolic and nutritional needs of the host. Previous studies of the metabolic demands of sickness have used model antigens, mitogens or pattern associated molecular patterns, which do not represent the full spectrum of response to a live pathogen infection. Thus, our study is the first of its kind to assess the full spectrum of metabolic, nutritional, immunological, and behavioral demands of live pathogen infection. MethodsMice were administered either a mock intraperitoneal (ip) injection of PBS (Control) or ip dose of Listeria and individually housed over the course of 12 days in Promethion metabolic cages to monitor their metabolic phenotype. In a parallel study, groups of mice were equivalently treated, yet conventionally housed and sacrificed at 3, 5, 7 and 10 days over the course of infection to determine splenic bacterial burden, Listeria-specific T cell response, and cellular metabolic status. ResultsWe observed that the period of the innate immune response (days 1–4) had the most metabolic demand, indicated by weight loss (P < 0.05), reduced activity (P < 0.05), increased sleep (P < 0.05), and decreased energy expenditure (P < 0.05). During the period of the adaptive immune response (days 5–10), there was little to no metabolic impact in the infected animals when compared to the uninfected control animals. We also observed increased GLUT1 expression (P < 0.05) on the membranes of myeloid cells during the period of highest metabolic demand, indicating shifts in cellular metabolism of innate immune cells during the early stages of infection. ConclusionsThe innate immune response is more metabolically taxing on the host compared to the adaptive immune response and places an increased metabolic demand on infected animals. Funding SourcesDepartmental startup funds to Elizabeth Hiltbold Schwartz.

Mechanotransduction, immunoregulation, and metabolic functions of CD31 in cardiovascular pathophysiology.

Biomechanical changes in the heart and vessels drive rapid and dynamic regulation of blood flow, a vital process for meeting the changing metabolic needs of the peripheral tissues at any given point in time. The fluid movement of the blood exerts haemodynamic stress upon the solid elements of the cardiovascular system: the heart, vessels, and cellular components of the blood. Cardiovascular diseases can lead to prolonged mechanical stress, such as cardiac remodelling during heart failure or vascular stiffening in atherosclerosis. This can lead to a significantly reduced or increasingly turbulent blood supply, inducing a shift in cellular metabolism that, amongst other effects, can trigger the release of reactive oxygen species and initiate a self-perpetuating cycle of inflammation and oxidative stress. CD31 is the most abundant constitutive co-signalling receptor glycoprotein on endothelial cells, which line the cardiovascular system and form the first-line of cellular contact with the blood. By associating with most endothelial receptors involved in mechanosensing, CD31 regulates the response to biomechanical stimuli. In addition, by relocating in the lipid rafts of endothelial cells as well as of cells stably interacting with the endothelium, including leucocytes and platelets, CD31-CD31 trans-homophilic engagement guides and restrains platelet and immune cell accumulation and activation and at sites of damage. In this way, CD31 is at the centre of mediating mechanical, metabolic, and immunological changes within the circulation and provides a single target that may have pleiotropic beneficial effects.

Activation of Peroxisome Proliferator-Activated Receptors α and δ Synergizes with Inflammatory Signals to Enhance Adoptive Cell Therapy.

Memory CD8+ T cells (Tmem) are superior mediators of adoptive cell therapy (ACT) compared with effector CD8+ T cells (Teff) due to increased persistence in vivo. Underpinning Tmem survival is a shift in cellular metabolism away from aerobic glycolysis towards fatty acid oxidation (FAO). Here we investigated the impact of the peroxisome proliferator-activated receptor (PPAR) agonist GW501516 (GW), an agent known to boost FAO in other tissues, on CD8+ T-cell metabolism, function, and efficacy in a murine ACT model. Via activation of both PPARα and PPARδ/β, GW treatment increased expression of carnitine palmitoyl transferase 1a, the rate-limiting enzyme of FAO, in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. T cells activated in the presence of GW and inflammatory signals, either mature dendritic cells or IL12, also demonstrated enhanced production of IFNγ and expression of T-bet. Despite high expression of T-bet, a characteristic of short-lived effector cells, GW-treated cells demonstrated enhanced persistence in vivo and superior efficacy in a model of ACT. Collectively, these data identify combined PPARα and PPARδ/β agonists as attractive candidates for further studies and rapid translation into clinical trials of ACT. SIGNIFICANCE: Dual activation of peroxisome proliferator-activated receptors α and δ improves the efficacy of adoptive cell therapy by reprogramming T-cell metabolism and cytokine expression.

TGF-β1 modifies histone acetylation and acetyl-coenzyme A metabolism in renal myofibroblasts.

Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine TGF-b1 on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Fibroblasts propagated from rat kidneys after ureteric obstruction were treated with recombinant TGF-b1 or vehicle for 48 hours. TGF-b1 -induced myofibroblast activation was accompanied by a net decrease in total H3 acetylation, although changes in individual marks were variable. This was paralleled by a generalised reduction in histone acetyltransferases (HAT), and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally this was manifest in a reduction in total HAT activity and increase in HDAC activity. TGF-b1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate or alternative sources of acetyl-CoA, without ameliorating changes in HAT/HDAC activity. In conclusion, TGF-b1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetylation, increased deacetylation and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-b1.

Can metabolomic profiling predict response to therapy?

Shifts in cellular metabolism are central to activation, differentiation and proliferation of inflammatory cells and can contribute to the pathogenesis of inflammatory diseases. Integrating metabolomics data with other omics data is a major challenge but might enable clinicians to stratify stages of disease and response to therapy in patients with rheumatoid arthritis.

Transcriptome profiling reveals the complexity of pirfenidone effects in idiopathic pulmonary fibrosis.

Despite the beneficial effects of pirfenidone in treating idiopathic pulmonary fibrosis (IPF), it remains unclear if lung fibroblasts (FB) are the main therapeutic target.To resolve this question, we employed a comparative transcriptomic approach and analysed lung homogenates (LH) and FB derived from IPF patients treated with or without pirfenidone.In FB, pirfenidone therapy predominantly affected growth and cell division pathways, indicating a major cellular metabolic shift. In LH samples, pirfenidone treatment was mostly associated with inflammation-related processes. In FB and LH, regulated genes were over-represented in the Gene Ontology node "extracellular matrix". We identified lower expression of cell migration-inducing and hyaluronan-binding protein (CEMIP) in both LH and FB from pirfenidone-treated IPF patients. Plasma levels of CEMIP were elevated in IPF patients compared to healthy controls and decreased after 7 months of pirfenidone treatment. CEMIP expression in FB was downregulated in a glioma-associated oncogene homologue-dependent manner and CEMIP silencing in IPF FB reduced collagen production and attenuated cell proliferation and migration.Cumulatively, our approach indicates that pirfenidone exerts beneficial effects via its action on multiple pathways in both FB and other pulmonary cells, through its ability to control extracellular matrix architecture and inflammatory reactions.

Abstract 5132: Lactic acidosis-mediated transcriptomic and metabolomic rewiring in pancreatic cancer cells

Abstract Tumor microenvironment plays a critical role in regulating tumor cell metabolism and progression. The tumor cells are constantly exposed to hypoxia, nutrient starvation, and extracellular acidification that contribute to tumor malignancy. These malignant cells exhibit Warburg effect by maintaining glycolytic phenotype even in the presence of oxygen and excrete lactate and protons via monocarboxylate transporters, and sodium-hydrogen antiporter 1 (NHE1) causing constant acidification of the tumor microenvironment. The tumor cells must thus adapt to acidosis to thrive in such hostile microenvironment. The purpose of the study is to identify the functional response of pancreatic cancer cells to the acidic tumor microenvironment. We performed integrated transcriptomic and metabolomic analyses to identify the existence of low pH-responsive transcriptional and metabolic regulators. Culturing the cells under low pH resulted in a reduction in cell growth and an increase in oxidative stress within the cells. RNA-seq-based transcriptomic analyses revealed that acidosis drives distinct gene expression patterns in PDAC cells with the expression of 499 and 496 genes up- and downregulated, respectively in PDAC cells. GSEA analyses of significantly modulated genes identified pathways including cell cycle, DNA repair, metabolic pathways, ABC transporters, glutathione metabolism, and genes involved in signaling cascades such as the inositide signaling pathway. As the transcriptomic analyses showed an enrichment of metabolic genes, intracellular metabolomic analyses revealed that chronic acidosis leads to a shift in cellular metabolism towards oxidative metabolism. Our studies demonstrate that while chronic acidosis in the tumor microenvironment leads to reduced proliferation and glycolysis in tumor cells, it also results in increased expression of genes involved in cellular migration, signaling pathways and switching to anaplerotic glutamine metabolism in response to an acidic tumor microenvironment. Overall, our studies demonstrate metabolic reprogramming in PDAC cells in response to low pH microenvironments creating a metabolic vulnerability that could be exploited for therapeutic targeting of pancreatic cancer. Citation Format: Divya Murthy, Jaime Abrego, Ryan James King, Pankaj Kumar Singh. Lactic acidosis-mediated transcriptomic and metabolomic rewiring in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5132.

HIF-2α is essential for carotid body development and function.

Mammalian adaptation to oxygen flux occurs at many levels, from shifts in cellular metabolism to physiological adaptations facilitated by the sympathetic nervous system and carotid body (CB). Interactions between differing forms of adaptive response to hypoxia, including transcriptional responses orchestrated by the Hypoxia Inducible transcription Factors (HIFs), are complex and clearly synergistic. We show here that there is an absolute developmental requirement for HIF-2α, one of the HIF isoforms, for growth and survival of oxygen sensitive glomus cells of the carotid body. The loss of these cells renders mice incapable of ventilatory responses to hypoxia, and this has striking effects on processes as diverse as arterial pressure regulation, exercise performance, and glucose homeostasis. We show that the expansion of the glomus cells is correlated with mTORC1 activation, and is functionally inhibited by rapamycin treatment. These findings demonstrate the central role played by HIF-2α in carotid body development, growth and function.

Abstract 444: Monocarboxylate transporter 1 inhibition with AZD3965 increases cancer cell dependence on bioenergetic metabolism predicating combination therapy with mitochondrial inhibitors

Abstract Monocarboxylate transporters (MCTs) are key mediators of lactate transport that have emerged as promising targets for anti-cancer therapy. The MCT1 inhibitor AZD3965 (AstraZeneca) has shown promising activity in various pre-clinical models and is currently in phase I/II clinical testing. Understanding the impact of this drug on tumour cell metabolism may unravel dependencies that could be exploited for combination therapy. Here we investigate changes in glucose metabolism induced by AZD3965 treatment using 13C NMR isotopomer analysis, and examine their significance for cell survival using mitochondrial metabolism inhibitors. Exposure of Raji human lymphoma cells to AZD3965 in media supplemented with [1-13C]glucose led to a marked reduction in glucose uptake and lactate production in the cellular growth media alongside a build-up in intracellular [3- 13C]lactate and [1-13C]glucose levels, indicative of blockade of lactate excretion and inhibition of overall glycolytic activity. These effects were concomitant with increased [4-13C]glutamate levels, consistent with re-routing of pyruvate towards mitochondrial metabolism and enhanced flux through oxidative pyruvate dehydrogenase. Further, AZD3965 treatment was paralleled with a significant increase in levels of steady state Krebs cycle-related metabolites (including succinate, fumarate, acetate and NAD+/NADH) and cellular ATP, as revealed by bioluminescent and 1H NMR analyses, indicating improved mitochondrial metabolism and cellular re-energization. Co-administration of the mitochondrial complex I inhibitor metformin or the mitochondrial pyruvate carrier inhibitor UK5099 markedly potentiated the anti-proliferative effects of AZD3965 and led to significantly increased cell death, indicating that the observed upregulation in mitochondrial metabolism was necessary to maintain cell survival under MCT1 inhibitor-induced metabolic stress. Similar effects were observed with a second human lymphoma cell line, Hut78. Our findings show that MCT1 inhibition leads to a shift in cellular metabolism towards mitochondrial bioenergetic metabolism, creating a metabolic vulnerability that could be exploited for combinatorial therapy to increase the efficacy of AZD3965. Citation Format: Mounia Beloueche-Babari, Teresa Casals Galobart, Slawomir Wantuch, Paul D. Smith, Martin O. Leach. Monocarboxylate transporter 1 inhibition with AZD3965 increases cancer cell dependence on bioenergetic metabolism predicating combination therapy with mitochondrial inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 444. doi:10.1158/1538-7445.AM2017-444

Characterization of the Pro-Inflammatory Cytokine IL-1β on Butyrate Oxidation in Colorectal Cancer Cells.

Cancer, in part, is driven, by alterations in cellular metabolism that promote cell survival and cell proliferation. Identifying factors that influence this shift in cellular metabolism in cancer cells is important. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that has been reported to be elevated in colorectal cancer patients. While much is known toward the effect of dietary nutrients on regulating inflammation and the inflammatory response, which includes cytokines such as IL-1β, far less is understood how cytokines impact nutrient fate to alter cancer cell metabolism. Butyrate, a nutrient derived from the fermentation of dietary fiber in the colon, is the preferential exogenous energetic substrate used by non-cancerous colonocytes, but is used less efficiently by colorectal cancer cells. To test whether IL-1β alters colonocyte energy metabolism, we measured butyrate oxidation in HCT116 colorectal cancer cells with and without IL-1β. We hypothesize that IL-1β will push cancerous colonocytes away from the utilization and oxidation of butyrate. In this study, we demonstrate that pretreatment of colorectal cancer cells with IL-1β diminished butyrate oxidation and NADH levels. This effect was blocked with the interleukin receptor antagonist A (IL-1RA). Moreover, IL-1β suppressed basal mitochondrial respiration and lowered the mitochondrial spare capacity. By using inhibitors to block downstream targets of the interleukin-1 receptor pathway, we show that p38 is required for the IL-1β-mediated decrease in butyrate oxidation. These data provide insight into the metabolic effects induced by IL-1β in colorectal cancer, and identify relevant targets that may be exploited to block the effects of this cytokine. J. Cell. Biochem. 118: 1614-1621, 2017. © 2016 Wiley Periodicals, Inc.

461 - Metabolic Plasticity Regulates Head and Neck Squamous Cell Cancer Growth-State Specific Oxidative Stress Response

Surgical resection followed by post-operative radiation is used as a standard treatment approach for locally advanced head and neck squamous cell cancer (HNSCC) patients. Unfortunately, even with aggressive approaches roughly 40% of patients with locoregionally advanced disease will ultimately succumb. 18F- Fluorothymidine Positron Emission Tomography (FLT PET) is a non-invasive imaging method that can be used in vivo to assess tumor proliferative status. Results showed that FLT PET successfully images HNSCC subjects with high lesion-to-background contrast. Surprisingly, our results also showed that HNSCC subjects with low FLT uptake (indicative of low proliferative index; LPI) in the primary tumor at base line have significantly poorer outcome compared to subjects with high tumor FLT uptake (indicative of high proliferative index; HPI). This intriguing clinical observation suggests that HNSCC growth-state significantly impacts therapy outcome. Consistent with this hypothesis, results from in vitro HNSCC cell culture studies showed that LPI-HNSCC cultures ( 20% S-phase cells). Radioresistance of LPI-HNSCC cultures is associated with a higher basal manganese superoxide dismutase activity, increases in radiation induced forkhead box M1 expression, suppression of late accumulation of reactive oxygen species (ROS), and absence of a radiation induced shift in cellular metabolism towards mitochondrial respiration. Interestingly, treatment with palmitate (substrate for the TCA cycle) increased oxygen consumption rate and ROS levels resulting in radiation sensitization of LPI-HNSCC cultures. These results support the hypothesis that metabolic plasticity regulates HNSCC growth-state specific radiation sensitivity.

Abstract B052: Peroxisome proliferator-activated receptor delta agonist GW501516 enhances the efficacy of adoptive cell therapy

Abstract Adoptive cell therapy (ACT) has demonstrated tremendous promise for the treatment of metastatic melanoma. Emerging evidence has indicated that memory CD8+ T cells (Tcm) are superior mediators of ACT than are effector CD8+ T cells (Teff) due to a superior ability to persist in vivo. Underpinning the survival advantage of Tcm is a shift in cellular metabolism away from aerobic glycolysis to instead favor fatty acid oxidation (FAO). This switch to FAO in Tcm requires the expression of carnitine palmitoyl transferase 1a (CPT1a), the rate-limiting enzyme of FAO. Here, we investigated the impact of the peroxisome proliferator-activated receptor (PPAR)δ/β agonist GW501516 (GW), an agent known to boost CPT1a expression and promote FAO in other tissues, on CD8+ T cell metabolism, function and performance in the murine B16-GP33 melanoma model. Via the activation of both PPARα and PPARδ/β we found that GW treatment increased CPT1a expression in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW treatment increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. Unexpectedly, we also found that T cells activated in the presence of GW as well as inflammatory signals, either LPS-matured dendritic cells (DCs) or IL-12, demonstrated enhanced production of interferon-γ. This increase in interferon-γ correlated with increased expression of the transcription factor T-bet. Despite the high T-bet expression, a characteristic of short-lived effector cells, GW-treated CD8+ T cells activated by LPS-treated DCs demonstrated an enhanced ability to persist in vivo. They also demonstrated improved performance in our murine model of ACT. Thus, our data indicates that combined treatment of CD8+ T cells with GW and inflammatory signals can result in in Teff cell with improved ability to produce cytokines but that have also shifted their metabolism to increase FAO akin to Tcm, allowing for enhanced in vivo persistence. These data identify GW501516, and potentially other combined PPARα and PPARδ/β agonists currently being developed, as attractive candidates for further studies and rapid translation into clinical trials of ACT. Citation Format: Samuel Saibil, Michael St. Paul, Pamela Ohahsi. Peroxisome proliferator-activated receptor delta agonist GW501516 enhances the efficacy of adoptive cell therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B052.

Diabetes propels the risk for cardiovascular disease: sweet monocytes becoming aggressive?

Diabetes strongly predisposes to cardiovascular disease (CVD), the leading cause of mortality in these patients, as well as in the entire population. Hyperglycemia is an important cardiovascular risk factor as shown by the observation that even transient periods of hyperglycemia, despite return to normoglycemia during follow-up, increase the risk for CVD, a phenomenon termed ‘hyperglycemic memory’. The molecular mechanisms underlying this phenomenon remain largely unknown. As inflammation plays an important role in the pathogenesis of atherosclerosis, we propose that long-term functional reprogramming of monocytes and macrophages, induced by hyperglycemia, plays an important role in the phenomenon of hyperglycemic memory leading to cardiovascular complications in patients with diabetes. In this review, we discuss recent insights showing that innate immune cells possess the capacity to reprogram their function through epigenetically mediated rewiring of gene transcription, a process termed ‘trained immunity’. The long-term reprogramming of monocytes can be induced by microbial as well as metabolic products, and involves a shift in cellular metabolism from oxidative phosphorylation to aerobic glycolysis. We hypothesize that hyperglycemia in diabetes patients induces long-term activation of monocytes and macrophages through similar mechanisms, thereby contributing to plaque development and subsequent macrovascular complications.

Abstract 1019: Investigation of cabozantinib, a MET and VEGFR2 inhibitor, on tumor metabolism and efficacy in a colorectal cancer patient-derived tumor explant model

Abstract Background: Anti-angiogenic therapy is commonly used for the treatment of metastatic colorectal cancer (mCRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. Upregulation of MET in response to anti-VEGF therapy may play an essential role in treatment resistance. Based on this premise, we investigated cabozantinib, an inhibitor of kinases including MET and VEGFR2, in mCRC patient-derived tumor explant (PDTX) models. Based on initial observations of anti-tumor activity, we hypothesized that cabozantinib may alter tumor metabolism concurrent with its antitumor effects, and compared its efficacy with regorafenib in our models. Material and Methods: Ten CRC PDTX models were treated with cabozantinib (30 mg/kg daily) or regorafenib (10 mg/kg daily) and treatment responses were determined after 28 days. The tumor growth inhibition index (TGII) was calculated to compare treatment effects on tumor growth between cabozantinib and regorafenib. RNA Seq was used to assess gene expression and pathways modulated by cabozantinib treatment. Proteins involved in metabolism and autophagy were evaluated by immunoblotting at day 3, 7 and 28. The effects of cabozantinib on tumor glucose metabolism were investigated by 18FDG-PET at baseline, 7 and 28 days following treatment. Lastly, combination effects of cabozantinib and an ULK1 inhibitor, an autophagy inhibitor, were evaluated on the HCT116 cell line by a clonogenic assay. Results: Cabozantinib (average TGII: 3.202) demonstrated antitumor effects among all 10 CRC explants that compared favorably to regorafenib (average TGII: 48.48). In addition, cabozantinib significantly reduced glucose uptake measured by 18FDG-PET at days 7 and 28. A comprehensive pathway analysis using RNA Seq post-cabozantinib treatment revealed a significant reduction in pyruvate metabolism and the TCA cycle in the most sensitive tumors. Protein analyses showed downregulation of hexokinase 1 and pyruvate dehydrogenase and a marked increase in many components of oxidative phosphorylation and autophagy (LC3 and Beclin) at day 7 and 28 following cabozantinib treatment. The combination of an ULK1 inhibitor and cabozantinib enhanced the activity of cabozantinib in the HCT116 CRC cell line. Conclusions: Cabozantinib showed significant antitumor activity compared to regorafenib in our CRC PDTX models. Alterations in glucose metabolism and autophagy were identified in cabozantinib sensitive tumors, suggesting that a shift in cellular metabolism facilitates the survival of tumor cells following treatment. The combination effect of cabozantinib and an ULK1 inhibitor supports the hypothesis that induction of autophagy may be a mechanism of cabozantinib resistance. These findings support further evaluation of cabozantinib in patients with mCRC. Acknowledgements: Exelixis for providing cabozantinib. Citation Format: Aaron J. Scott, Rachel Yahn, Stacey Bagby, Kendra Huber, Natalie Serkova, Wells Messersmith, John Arcaroli. Investigation of cabozantinib, a MET and VEGFR2 inhibitor, on tumor metabolism and efficacy in a colorectal cancer patient-derived tumor explant model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1019.

Oxidative and nitrosative signalling in pulmonary arterial hypertension - Implications for development of novel therapies.

Pulmonary arterial hypertension (PAH) is a syndrome characterised by an increase in pulmonary vascular resistance. This results in elevated resting pulmonary artery pressure and leads to progressive right ventricular (RV) failure, secondary to increased afterload. Although initially thought to be a disease driven primarily by endothelial dysfunction with a resultant vasoconstrictor versus vasodilator imbalance, it has become increasingly apparent that the rise in pulmonary vascular resistance that causes RV failure is also attributable to pulmonary vascular remodelling. This inflammatory, hyper-proliferative and anti-apoptotic phenotype is accompanied by a metabolic switch from physiological mitochondrial oxidative phosphorylation to aerobic glycolysis. The molecular pathways triggering this cellular metabolic shift have been the subject of extensive investigation, as their discovery will inevitably lead to new therapeutic targets. Reactive oxygen/nitrogen species (ROS/RNS) including hydrogen peroxide, superoxide and peroxynitrite are second messenger molecules that are involved in functional oxidative and nitrosative modification of proteins. Dysregulation of oxidative signalling caused by an excess of ROS and RNS relative to antioxidants has been heavily implicated in the underlying pathophysiology of PAH and likely participates in this metabolic reprogramming. This review will focus on the role of oxidative signalling and redox reactions to the molecular pathology of PAH. In addition, promising novel therapeutic agents targeting these pathways will be discussed.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Antisense-RNA-Mediated Gene Downregulation in Clostridium pasteurianum

Clostridium pasteurianum is receiving growing attention for its unique metabolic properties, particularly its ability to convert waste glycerol and glycerol-rich byproducts into butanol, a prospective biofuel. Genetic tool development and whole genome sequencing have recently been investigated to advance the genetic tractability of this potential industrial host. Nevertheless, methodologies for tuning gene expression through plasmid-borne expression and chromosomal gene downregulation are still absent. Here we demonstrate plasmid-borne heterologous gene expression and gene knockdown using antisense RNA in C. pasteurianum. We first employed a common thermophilic β-galactosidase (lacZ) gene reporter system from Thermoanaerobacterium thermosulfurogenes to characterize two promoters involved in the central fermentative metabolism of C. pasteurianum. Due to a higher level of constitutive lacZ expression compared to the ferredoxin gene (fdx) promoter, the thiolase (thl) promoter was selected to drive expression of asRNA. Expression of a lacZ asRNA resulted in 52%–58% downregulation of β-galactosidase activity compared to the control strain throughout the duration of culture growth. Subsequent implementation of our asRNA approach for downregulation of the native hydrogenase I gene (hydA) in C. pasteurianum resulted in altered end product distribution, characterized by an increase in production of reduced metabolites, particularly butyrate (40% increase) and ethanol (25% increase). Knockdown of hydA was also accompanied by increased acetate formation and lower levels of 1,3-propanediol, signifying a dramatic shift in cellular metabolism in response to inhibition of the hydrogenase enzyme. The methodologies described herein for plasmid-based heterologous gene expression and antisense-RNA-mediated gene knockdown should promote rational metabolic engineering of C. pasteurianum for enhanced production of butanol as a prospective biofuel.

Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment

Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots.From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants.

Expansion of Primary Human AML by Aryl Hydrocarbon Receptor Antagonism Minimally Affects Leukemic Transcriptional Profiles but Alters Cellular Metabolism

Small molecule-based antagonism of the aryl hydrocarbon receptor (AHR) by StemRegenin1 (SR1) promotes ex vivo expansion and maintenance of primary human hematopoietic stem cells (HSCs) as well as acute myeloid leukemia (AML) cells. However, basis and nature of SR1 induced expansion of human AML remains unknown. Here, global expression profiling on 7 clinically diverse human AML patient samples treated ex vivo with a synthesized analog of SR1 (aSR1) uncovered that only as few as 750 genes were differentially regulated. Uniquely, aSR1 treatment did not modulate self-renewal associated pathways including Hedgehog, Notch or Wnt across patient samples, but instead resulted in overall upregulation of the oxidative phosphorylation metabolic pathway. Higher oxygen consumption rates, along with increased sensitivity to the chemotherapeutic agent cytarabine (AraC) validated that aSR1-induced transcriptional profiles lead to functional enhancement of oxidative phosphorylation. Our study reveals that aSR1 induces minor alterations to the leukemic transcriptional profile leading to a shift in cellular metabolism. This finding should further instruct use of SR1-mediated expansion for mechanistic studies of leukemic self-renewal and the development of drug screening platforms using patient specific AML samples.

Apoptolidins A and C activate AMPK in metabolically sensitive cell types and are mechanistically distinct from oligomycin A

Apoptolidin A was first isolated as a secondary metabolite of a Nocardiopsis sp. and is the founding member of a family of potential selective cancer cell toxins. We now report the isolation, production and pharmacological characterization of apoptolidins A and C from an alternate actinomycete producer, an Amycolatopsis sp. from soil samples collected in Indonesia. We investigated the action of apoptolidins A and C in representative human glioblastoma cells, lung cancer cells and mouse embryonic fibroblasts (MEFs) to better understand the mechanism of action of the known apoptolidins. Shifts in cellular metabolism in intact cells and the status of the AMP-activated protein kinase (AMPK) stress pathway in response to apoptolidin A were entirely consistent with the actions of an ATP synthase inhibitor. We find the metabolic phenotype of the cell to be a critical determinant of apoptolidin sensitivity and the likely basis for cancer cell selectivity. The apoptolidins induce indirect activation of AMPK and trigger autophagy in sensitive cell types without significant inhibition of mTORC1. Human U87-MG glioblastoma cells and wild type MEFs showed increased phosphorylation of AMPK (Thr172), ACC (Ser79) and ULK1 (Ser555), whereas AMPKα-null MEFs and more glycolytic SF-295 glioblastoma cells lacked this response. Although both are reported to be selective inhibitors of mitochondrial ATP synthase, differences between apoptolidin- and oligomycin A-induced responses in cells indicate that the action of these macrolides is not identical.