Metabolic flux analysis of neural cell metabolism in primary cultures

Dissertation presented to obtain the Ph.D degree in Biochemistry, Neuroscience

Insights into neuronal cell metabolism using NMR spectroscopy: uridyl diphosphate N-acetyl-glucosamine as a unique metabolic marker.

Making the switch: Compounds 1 and 2 are used as metabolic markers for NMR detection. When neuronal cells switch to a glycolytic state, an uneven distribution of (13) C in the N-acetyl group results, thus giving a mixture of the metabolites 1 and 2. It is therefore possible to monitor flux through different metabolic pathways, such as glycolysis, the tricarboxylic acid cycle, and the hexosamine biosynthetic pathway, using a single molecule.

Targeting cancer cell metabolism in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer death by 2030. Current therapeutic options are limited, warranting an urgent need to explore innovative treatment strategies. Due to specific microenvironment constraints including an extensive desmoplastic stroma reaction, PDAC faces major metabolic challenges, principally hypoxia and nutrient deprivation. Their connection with oncogenic alterations such as KRAS mutations has brought metabolic reprogramming to the forefront of PDAC therapeutic research. The Warburg effect, glutamine addiction, and autophagy stand as the most important adaptive metabolic mechanisms of cancer cells themselves, however metabolic reprogramming is also an important feature of the tumor microenvironment, having a major impact on epigenetic reprogramming and tumor cell interactions with its complex stroma. We present a comprehensive overview of the main metabolic adaptations contributing to PDAC development and progression. A review of current and future therapies targeting this range of metabolic pathways is provided.

A Multivariate Calibration Procedure for UV/VIS Spectrometric Monitoring of BHK-21 Cell Metabolism and Growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electrochemical imaging of living cell metabolism: investigation on Warburg effect in cancer

Cancer is one of the principal causes of death in the world; almost 8.2 million of deaths were counted in 2012. Emerging evidences indicate that most of the tumors have an increased glycolytic rate and a detriment of oxidative phosphorylation to support abnormal cell proliferation; this phenomenon is known as aerobic glycolysis or Warburg effect. This switching toward glycolysis implies that cancer tissues metabolize approximately tenfold more glucose to lactate in a given time and the amount of lactate released from cancer tissues is much greater than from normal ones. In view of these fundamental discoveries alterations of the cellular metabolism should be considered a crucial hallmark of cancer. Therefore, the investigation of the metabolic differences between normal and transformed cells is important in cancer research and it might find clinical applications. The aim of the project was to investigate the cellular metabolic alterations at single cell level, by monitoring glucose and lactate, in order to provide a better insight in cancer research. For this purpose, electrochemical techniques have been applied. Enzyme-based electrode biosensors for lactate and glucose were –ad hoc- optimized within the project and used as probes for Scanning Electrochemical Microscopy (SECM). The UME biosensor manufacturing and optimization represented a consistent part of the work and a full description of the sensor preparation protocols and of the characterization methods employed is reported. This set-up (SECM used with microbiosensor probes) enabled the non-invasive study of cellular metabolism at single cell level. The knowledge of cancer cell metabolism is required to design more efficient treatment strategies.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Functional analysis of anionic phospholipids in cell metabolism in {\it Escherichia coli\/}

The major goal of this work was to understand the function of anionic phospholipid in E. coli cell metabolism. One important finding from this work is the requirement of anionic phospholipid for the DnaA protein-dependent initiation of DNA replication. An rnhA mutation, which bypasses the need for the DnaA protein through induction of constitutive stable DNA replication, suppressed the growth arrest phenotype of a $pgsA$ mutant in which the synthesis of anionic phospholipid was blocked. The maintenance of plasmids dependent on an $oriC$ site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein. In addition, structural and functional requirements of two major anionic phospholipids, phosphatidylglycerol and cardiolipin, were examined. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for the naturally occurring phosphatidylglycerol. The requirement for phosphatidylglycerol was concluded to be more than maintenance of the proper membrane surface charge. Examination of the role of cardiolipin revealed its ability to replace the zwitterionic phospholipid, phosphatidylethanolamine, in maintaining an optimal membrane lipid organization. This work also reported the DNA sequence of the cls gene, which encodes the CL synthase responsible for the synthesis of cardiolipin. ^

Latent and Lytic KSHV Infection Require Host Cell Metabolism

Thesis (Ph.D.)--University of Washington, 2016-06

Modeling hybridoma cell metabolism using a generic genome-scale metabolic model of Mus musculus

The reconstructed cellular metabolic network of Mus musculus, based on annotated genomic data, pathway databases, and currently available biochemical and physiological information, is presented. Although incomplete, it represents the first attempt to collect and characterize the metabolic network of a mammalian cell on the basis of genomic data. The reaction network is generic in nature and attempts to capture the carbon, energy, and nitrogen metabolism of the cell. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, including transport reactions between the compartments and the extracellular medium. The reaction list consists of 872 internal metabolites involved in a total of 1220 reactions, whereof 473 relate to known open reading frames. Initial in silico analysis of the reconstructed model is presented.

The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

Ions and Small Molecules as Modulators of F1FO-ATPase, Mitochondrial Bioenergetics and Cell Metabolism

The properties of the mitochondrial F1FO-ATPase activated by the natural cofactor Mg2+ or by Ca2+, were studied, mainly on heart mitochondria from swine, widely used in translational medicine. The Ca2+ driven conformational changes in the F1FO-ATPase form the mitochondrial permeability transition pore (mPTP), which triggers regulated cell death and is involved in severe pathologies. The Ca2+-activated F1FO-ATPase hydrolyzes ATP with kinetics slightly different from those of the Mg2+-ATPase. Known F1-ATPase inhibitors inhibit both the Ca2+-activated F1FO-ATPase and the mPTP formation strengthening the molecular link between them. The different Gd3+ effects on the Ca2+- and Mg2+-activated F1FO-ATPases confirm their difference as also phenylglyoxal which preferentially inhibits the Ca2+-activated F1FO-ATPase. The effects of phenylarsine and dibromobimane, which interact with differently distant Cys thiols, show that mPTP opening is ruled by nearby or distant dithiols. Bergamot polyphenols and melatonin inhibit the mPTP and ROS formation. H2S, a known cardiovascular protector, unaffects the F1FO-ATPase, but inhibits Ca2+ absorption and indirectly the mPTP, both in swine heart and mussel midgut gland mitochondria. New generation triazoles inhibit the Ca2+-activated F1FO-ATPase and the mPTP, but unaffect the Mg2+-activated F1FOATPase. In parallel, the energy metabolism was investigated in mammalian cells. In boar sperm ATP is mainly produced by mitochondrial oxidative phosphorylation (OXPHOS), even if it decreases over time because of less active mitochondria. Insufficient ATP may induce sperm dysfunction. Also, canine mesenchymal stem cells rely on OXPHOS; those from umbilical cord which produce more ATP than those from adipose tissue, seem preferable for transplant studies. The intestinal porcine enterocyte cell line IPEC-J2, used for human gut research, responds to different fetal bovine serum concentrations by remodeling OXPHOS without altering the bioenergetic parameters. The IPEC-J2 bioenergetics is modulated by Vitamin K vitamers. These data shoulder cell bioenergetics as precious tool for medical research.

Re-thinking cell cycle regulators: the cross-talk with metabolism.

Analysis of genetically engineered mice deficient in cell cycle regulators, including E2F1, cdk4, and pRB, showed that the major phenotypes are metabolic perturbations. These key cell cycle regulators contribute to lipid synthesis, glucose production, insulin secretion, and glycolytic metabolism. It has been shown that deregulation of these pathways can lead to metabolic perturbations and related metabolic diseases, such as obesity and type II diabetes. The cyclin-cdk-Rb-E2F1 pathway regulates adipogenesis in addition to its well-described roles in cell cycle regulation and cancer. It was also shown that E2F1 directly participates in the regulation of pancreatic growth and function. Similarly, cyclin D3, cdk4, and cdk9 are also adipogenic factors with strong effects on whole organism metabolism. These examples support the emerging notion that cell cycle regulatory proteins also modulate metabolic processes. These cell cycle regulators are activated by insulin and glucose, even in non-proliferating cells. Most importantly, these cell cycle regulators trigger the adaptive metabolic switch that normal and cancer cells require in order to proliferate. These changes include increased lipid synthesis, decreased oxidative metabolism, and increased glycolytic metabolism. In summary, these factors are essential regulators of anabolic biosynthetic processes, blocking at the same time oxidative and catabolic pathways, which is reminiscent of cancer cell metabolism.

Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells.