Deciphering the Role of LRH-1 in Liver Intermediary Metabolism and Cancer

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Gaining insight into the molecular pathways involved in the development of HCC is hence a prerequisite to design and develop novel and effective therapeutic strategies. Metabolic reprogramming is a universal feature of tumor cells and is characterized by increased glycolysis and diminished oxidative phosphorylation, a phenomenon known as the Warburg effect. More recently, it has become evident that tumor cells heavily rely on gluta-mine metabolism to compensate for the Warburg effect and to replenish the tricarboxylic acid (TCA) cycle. However, the molecular mechanisms by which glutamine supports tumor cell metabolism remain largely unexplored. In this thesis, I have established the role of the nuclear receptor LRH-1 as a key regulator in the process of hepatic tumorigenesis. I observed that LRH-1 promotes DEN-induced hepatocellular carcinogenesis (HCC). I demonstrated that LRH-1 facilitates the production of NADPH from glutamine by favoring a non-canonical glutamine pathway that optimizes reductive biosynthesis. Importantly, chronic and acute disrup-tion of LRH-1 also impaired glutamine-induced anaplerosis and Î±-KG availability, ultimately leading to im-paired mTORC1 signaling to block cell proliferation. Moreover, LRH-1 also coordinates glutamine-dependent asparagine synthesis to protect tumor cell from apoptosis induced by glutamine depletion itself. As a result, gain-of-function of LRH-1 sustained aspar-agine homeostasis upon glutamine-deprivation to promote cell survival. Collectively, these studies unveiled an unexpected role of LRH-1 in cancer intermediary metabolism, and warranted further studies on pharma-cological inhibition of LRH-1 to interfere with hepatocellular carcinogenesis.

GLUT1 and GLUT3 in non-small cell lung cancer

Mark Jonathan Masin

While altered metabolism is a well-established tumor cell trait, the specifics of metabolic rewiring during cancer development and progression are yet to be fully understood. The Warburg effect, or aerobic glycolysis, has been described in a wide array of malignancies and has long been considered as an inefficient mean to fill energetic requirements. Emerging evidence however supports a role that extends beyond that, with glycolytic intermediates diverting to anabolic pathways to allow growth and proliferation. Importantly, the first and most critical rate-limiting step in glycolysis is glucose uptake. Fittingly, high affinity, high capacity glucose transporters such as GLUT1 and to a lesser extent GLUT3, are upregulated in cancer. Given the centrality of glycolysis in tumor biology and reported clinical observations relating to glucose transporter expression patterns, the present work focuses on characterizing the role of GLUT1 and GLUT3 in the context of non-small cell lung cancer (NSCLC), the most prevalent histological subtype of lung cancer. Linking glucose metabolism and a program involved in early steps of metastasis, part of the findings uncovers an aberrant expression of GLUT3 as integral to the epithelial-mesenchymal transition (EMT) in NSCLC. Indeed, a unique association between GLUT3 and a mesenchymal status is observed in a panel of human NSCLC cell lines. Furthermore, GLUT3 expression is increased during EMT by a mechanism that involves direct binding of EMT mediating transcription factor ZEB1 to the GLUT3 (SLC2A3) gene. Data also supports a functional role of GLUT3 in proliferation, as inhibiting GLUT3 expression reduces glucose import and proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. In an analysis of a large microarray data collection of human NSCLC samples, GLUT3 expression is found to correlate with EMT markers, and is prognostic of poor overall survival. Taken collectively, the data reveal an important role for GLUT3 in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Owing to the restricted expression of this transporter in healthy individuals, its presence in lung tumor cells may represent a noteworthy prospect for targeted therapy. The contrastingly ubiquitous nature of GLUT1 in normal and transformed tissue warrants in vivo investigation to determine expression patterns and contribution tumor development. To that end, generation of autochthonous lung cancer mouse models allows assessment of the effect of tumor exclusive GLUT1 loss. Ensuing preliminary results allude to a possible role of GLUT1 in specific histological lesions. The significance of this specificity is currently under investigation.

EPFL2015

Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures

Johan Auwerx, Richardus Houtkooper, Cyril Mikhail

Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish.

Soc Neuroscience2012

Mathematical Modeling of Brain Energy Metabolism, Measured with PET and MRS in Rodents

Bernard Lanz

Positron emission tomography (PET) and nuclear magnetic resonance spectroscopy (MRS) are two biomedical measurement techniques developed in the end of the XXth century, which drastically improved the amount of accessible information available in vivo. PET became popular through the most widely used tracer, fluorodeoxyglucose (FDG), which enables the measurement of the local glucose utilization and is nowadays routinely applied in clinical practice. Nuclear magnetic resonance is used in a large array of applications such as in analytical chemistry or chemical structure determination and is essentially known for its versatile medical imaging capabilities, grouped under the name of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). In vivo MRS measures concentrations of metabolites by interacting with various nuclei such as 1H, 13C or 15N, with the major asset that MRS enables the identification of the chemical position at which the detected nuclei is located in the measured molecule. When coupled with labeled substrate infusions, dynamic MRS gives the opportunity to probe specific biochemical reactions directly in vivo, which opens the way to a wide range of metabolic studies. However, the correct interpretation of the elaborate dynamic labeling data acquired either with MRS or PET tracer experiments requires the application of adapted metabolic models to derive quantitative metabolic rates characterizing the biochemical processes under study. The work of this thesis involves both PET and MRS studies of brain energy metabolism in rodents and focuses on the development of adapted metabolic models to derive reliable metabolic fluxes characterizing various brain metabolic processes, such as glucose consumption, glial and neuronal oxidative metabolism and neurotransmission. FDG-PET enables the measurement of the cerebral metabolic rate of glucose (CMRGlc) using a three-compartment metabolic model for FDG transport across the blood-brain barrier and FDG phosphorylation to FDG-6P. Although this method is well established, a main drawback of CMRGlc measurement with FDG-PET is the necessity to characterize the available FDG concentration in the plasma over the experiment duration, the so-called arterial input function. This constraint is a strong limitation for preclinical studies in rodents, due to their low blood volume. We developed therefore a new method to extract the FDG input function directly from the PET images in rats and mice, using the time-activity curve of a voxel located in the inferior vena cava. The method was validated by comparison with a dedicated external blood counter and manual blood sampling. Using this method, a CMRGlc of ~0.22 μmol/g/min was determined in the mouse cortex. Glial oxidative metabolism can be specifically assessed using glial specific substrates, in particular [1-11C] acetate. However, no existing metabolic model described the 11C tissue activity curve in terms of neuroglial energy metabolism. In order to extract quantitative metabolic fluxes characterizing the glial TCA cycle rate and glutamate/glutamine cycling, we adapted the neuroglial two-compartment modeling approach previously used in 13C MRS cerebral metabolic studies to interpret positron emission data following 11C-acetate injection in the rat. The precision and accuracy of the estimated metabolic parameters was tested and a composite glial metabolic flux Vgtg=0.136 ± 0.042 μmol/g/min and a neurotransmission flux Vnt=0.170 ± 0.103 μmol/g/min were obtained. This approach enabled a direct comparison of the metabolic fluxes measured using 11C PET with the values determined with 13C MRS. In the field of 13C MRS, we took advantage of the higher sensitivity and spectral resolution available at high magnetic field (14.1T) to measure glial and neuronal oxidative metabolism, glutamatergic neurotransmission and pyruvate carboxylation in more details and with higher precision using both [1,6-13C2] glucose and [2-13C] acetate infusions in rats. In the acetate study, the glial specific uptake of acetate enabled a quantification of glial oxidative metabolism with unrivalled precision. We could estimate for the first time separately the transmitochondrial flux Vx in the glial and neuronal compartment, a biochemical pathway which has been subject of strong controversy. The glial and neuronal Vx were determined with high precision, as calculated through Monte Carlo simulation, and their value was on the same order of magnitude than the respective glial and neuronal TCA cycle flux. The model was extended to estimate the fraction of glutamate located in the glial compartment. A glial glutamate concentration of 0.6 ± 0.1 μmol/g was found. The values of the different metabolic fluxes characterizing the neuroglial system (Vtcag=0.27, Vxg=0.17, VPC=0.087, VNT=0.15, Vtcan=0.37 and Vxn=0.46 μmol/g/min) were in very good agreement with the values found using [1,6-13C2] glucose infusion. In addition, the effect of the MRS temporal resolution, experiment duration and signal- to-noise ratio on the precision of the derived metabolic fluxes was analyzed. Previous simulation studies showed that two-compartment modeling of neuroglial metabolism using 13C glucose infusion could lead to unreliable parameter estimations. However, using polarization transfer 13C MRS at 14.1T, the carbon positions C4, C3 and C2 of glutamate and glutamine as well as the positions C3 and C2 of aspartate could be measured with high SNR. The C2 positions were used in two-compartment modeling for the first time, enabling a precise measurement of the apparent glutamatergic neurotransmission and glial metabolism, including pyruvate carboxylation, as confirmed by Monte Carlo simulation. The glial dilution at the level of acetyl-CoA, which is related to the metabolism of other substrates than glucose, could also be characterized. Brain metabolism under hyperammonemic conditions was probed with 15N MRS under labeled ammonia infusion and modeled using a modified neuroglial two- compartment model describing the dynamics of [5-15N] glutamine, [2-15N] glutamate and [2-15N] glutamine. Due to detoxification processes, the total glutamine concentration, measured with 1H MRS, was steadily increasing. This non-steady- state metabolic condition resulting from the hyperammonemic stress was taken into account in the model, resulting in the determination of the apparent neurotransmission (0.26 ± 0.030 μmol/g/min), the glutamate dehydrogenase (0.029 ± 0.002 μmol/g/min) and net glutamine accumulation (0.033 ± 0.001 μmol/g/min). Finally, compartmental modeling was applied to 13C labeling studies in the awake rat, fed during 5, 24 or 48 h with a [1-13C]-labeled glucose solution, to determine brain glycogen and NAA turnover times (τGlyc=5.3 ± 3.2 h and τNAA=15.6 ± 6.5 h), using 13C spectra of brain extracts. A group of rats followed a mild brain activation protocol over 5 hours, which resulted in a decreased glycogen turnover time (τGlyc =2.9 ± 1.2 h).