88 research outputs found
HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism
Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxiainducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis
Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-13C6]-d-glucose tracer in mice
Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal 13C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-13C]-d-glucose. 13C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional 13C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. 13C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of 13C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host
Mitochondrial Disease in Autism Spectrum Disorder Patients: A Cohort Analysis
Previous reports indicate an association between autism spectrum disorders (ASD) and disorders of mitochondrial oxidative phosphorylation. One study suggested that children with both diagnoses are clinically indistinguishable from children with idiopathic autism. There are, however, no detailed analyses of the clinical and laboratory findings in a large cohort of these children. Therefore, we undertook a comprehensive review of patients with ASD and a mitochondrial disorder.We reviewed medical records of 25 patients with a primary diagnosis of ASD by DSM-IV-TR criteria, later determined to have enzyme- or mutation-defined mitochondrial electron transport chain (ETC) dysfunction. Twenty-four of 25 patients had one or more major clinical abnormalities uncommon in idiopathic autism. Twenty-one patients had histories of significant non-neurological medical problems. Nineteen patients exhibited constitutional symptoms, especially excessive fatigability. Fifteen patients had abnormal neurological findings. Unusual developmental phenotypes included marked delay in early gross motor milestones (32%) and unusual patterns of regression (40%). Levels of blood lactate, plasma alanine, and serum ALT and/or AST were increased at least once in 76%, 36%, and 52% of patients, respectively. The most common ETC disorders were deficiencies of complex I (64%) and complex III (20%). Two patients had rare mtDNA mutations of likely pathogenicity.Although all patients' initial diagnosis was idiopathic autism, careful clinical and biochemical assessment identified clinical findings that differentiated them from children with idiopathic autism. These and prior data suggest a disturbance of mitochondrial energy production as an underlying pathophysiological mechanism in a subset of individuals with autism
Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production
Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches
Inter-laboratory comparison of nanoparticle size measurements using dynamic light scattering and differential centrifugal sedimentation
Nanoparticle in vitro toxicity studies often report contradictory results with one main reason being insufficient material characterization. In particular the characterization of nanoparticles in biological media remains challenging. Our aim was to provide robust protocols for two of the most commonly applied techniques for particle sizing, i.e. dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) that should be readily applicable also for users not specialized in nanoparticle physico-chemical characterization. A large number of participants (40, although not all participated in all rounds) were recruited for a series of inter-laboratory comparison (ILC) studies covering many different instrument types, commercial and custom-built, as another possible source of variation. ILCs were organized in a consecutive manner starting with dispersions in water employing well-characterized near-spherical silica nanoparticles (nominal 19 nm and 100 nm diameter) and two types of functionalized spherical polystyrene nanoparticles (nominal 50 nm diameter). At first each laboratory used their in-house established procedures. In particular for the 19 nm silica particles, the reproducibility of the methods was unacceptably high (reported results were between 10 nm and 50 nm). When comparing the results of the first ILC round it was observed that the DCS methods performed significantly worse than the DLS methods, thus emphasizing the need for standard operating procedures (SOPs). SOPs have been developed by four expert laboratories but were tested for robustness by a larger number of independent users in a second ILC (11 for DLS and 4 for DCS). In a similar approach another SOP for complex biological fluids, i.e. cell culture medium containing serum was developed, again confirmed via an ILC with 8 participating laboratories. Our study confirms that well-established and fit-for-purpose SOPs are indispensable for obtaining reliable and comparable particle size data. Our results also show that these SOPs must be optimized with respect to the intended measurement system (e.g. particle size technique, type of dispersant) and that they must be sufficiently detailed (e.g. avoiding ambiguity regarding measurand definition, etc.). SOPs may be developed by a small number of expert laboratories but for their widespread applicability they need to be verified by a larger number of laboratories
Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy
Background: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1alpha (HIF-1alpha) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways
Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences
Efficient processing of information by the central nervous system (CNS) represents an important evolutionary advantage. Thus, homeostatic mechanisms have developed that provide appropriate circumstances for neuronal signaling, including a highly controlled and stable microenvironment. To provide such a milieu for neurons, extracellular fluids of the CNS are separated from the changeable environment of blood at three major interfaces: at the brain capillaries by the blood-brain barrier (BBB), which is localized at the level of the endothelial cells and separates brain interstitial fluid (ISF) from blood; at the epithelial layer of four choroid plexuses, the blood-cerebrospinal fluid (CSF) barrier (BCSFB), which separates CSF from the CP ISF, and at the arachnoid barrier. The two barriers that represent the largest interface between blood and brain extracellular fluids, the BBB and the BCSFB, prevent the free paracellular diffusion of polar molecules by complex morphological features, including tight junctions (TJs) that interconnect the endothelial and epithelial cells, respectively. The first part of this review focuses on the molecular biology of TJs and adherens junctions in the brain capillary endothelial cells and in the CP epithelial cells. However, normal function of the CNS depends on a constant supply of essential molecules, like glucose and amino acids from the blood, exchange of electrolytes between brain extracellular fluids and blood, as well as on efficient removal of metabolic waste products and excess neurotransmitters from the brain ISF. Therefore, a number of specific transport proteins are expressed in brain capillary endothelial cells and CP epithelial cells that provide transport of nutrients and ions into the CNS and removal of waste products and ions from the CSF. The second part of this review concentrates on the molecular biology of various solute carrier (SLC) transport proteins at those two barriers and underlines differences in their expression between the two barriers. Also, many blood-borne molecules and xenobiotics can diffuse into brain ISF and then into neuronal membranes due to their physicochemical properties. Entry of these compounds could be detrimental for neural transmission and signalling. Thus, BBB and BCSFB express transport proteins that actively restrict entry of lipophilic and amphipathic substances from blood and/or remove those molecules from the brain extracellular fluids. The third part of this review concentrates on the molecular biology of ATP-binding cassette (ABC)-transporters and those SLC transporters that are involved in efflux transport of xenobiotics, their expression at the BBB and BCSFB and differences in expression in the two major blood-brain interfaces. In addition, transport and diffusion of ions by the BBB and CP epithelium are involved in the formation of fluid, the ISF and CSF, respectively, so the last part of this review discusses molecular biology of ion transporters/exchangers and ion channels in the brain endothelial and CP epithelial cells
Elemental changes in the hippocampal formation following two different formulas of ketogenic diet: an X-ray fluorescence microscopy study
SEM/EDS i mikro-spektroskopia Ramana w analizie włókien polipropylenowych modyfikowanych ditlenkiem tytanu
In order to impart photo-catalytic properties to fibres, one can modify them by means of titanium dioxide. Such a modification should provide the best effectiveness with no deterioration in the basic properties of the fibres modified. Accordingly the modifiers should be deposited on the surface of fibres instead of adding them to the whole mass of the fibreforming polymer. To characterise polypropylene fibres (PP) modified with titanium dioxide doped with nano silver (TiO2/Ag), Raman micro-spectroscopy and SEM/EDS microanalysis were used. The combination of both techniques in the analysis of the fibre surface and cross-section allows qualitative and structural assessment of the distribution of titania modifier in the fibre.Do nadawania włóknom funkcji fotokatalitycznych stosuje się modyfikacje ditlenkiem tytanu. Modyfikacja taka powinna zapewnić najlepszą efektywność fotokatalityczną bez pogorszenia podstawowych właściwości modyfikowanych włókien. W związku z tym modyfikator powinien znajdować się na powierzchni włókien, a nie w całej objętości polimeru włóknotwórczego. Do oceny efektu modyfikacji włókien polipropylenowych (PP) ditlenkiem tytanu domieszkowanym srebrem (TiO2/Ag) zastosowano techniki mikro-spektroskopii Ramana oraz elektronowej mikroskopii skaningowej połączonej z mikroanalizą rentgenowską SEM/EDS. Połączenie obu technik do analizy powierzchni oraz przekroju poprzecznego włókien (PP) pozwoliło na ocenę jakościowego i strukturalnego efektu modyfikacji
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