Hyperpolarized Magnetic Resonance Methods to Investigate Metabolic Pathways in Liver and Immune Cells

Dysregulation of cellular metabolism is a key feature of major diseases such as diabetes and cancer. Monitoring cellular metabolic alterations is of critical importance for improved understanding of disease progression and the development of novel therapeutics. Carbon-13 magnetic resonance spectroscopy (13C MRS), which can detect and quantitate metabolites in a non-invasive manner, is well suited for clinical and pre-clinical metabolic studies. The low natural abundance of 13C allows one to trace exogenously administered 13C-labeled substrates by MRS and which enables the study of complex metabolic pathways in living subjects. However, the low sensitivity of 13C MRS limits the number of metabolites that can be detected with this technique. Hyperpolarized (HP) 13C MRS using dissolution dynamic nuclear polarization (d-DNP) is a novel metabolic imaging technique which allows real-time monitoring of the dynamics of in vivo metabolism by temporarily increasing the signal intensity more than 10,000-fold over conventional 13C MRS. This thesis is focused on the biomedical applications of HP 13C MRS, particularly to investigate in vivo hepatocellular energy metabolism in rodents and metabolic changes in active immune cells. Customized MRI/NMR probes and associated acquisition sequences were designed and implemented to improve the sensitivity and throughput of the HP MR experiments. A HP 13C MRS protocol for the sensitive in vivo detection of hepatic pyruvate metabolism was developed. HP [1-13C]pyruvate was infused at progressively decreasing doses to healthy rats under different nutritional conditions via a rapid automated infusion. It was shown that liver metabolism can be measured in vivo with HP [1-13C]pyruvate administered at near-physiological levels. While conversion to HP bicarbonate was detected in the liver of fasted rats, treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinate resulted in significantly lower levels compared to fed rats, which supports the notion that hepatic gluconeogenic metabolism can be directly probed in vivo with HP pyruvate. Additionally, real-time in vivo hepatocellular glutamine and pyruvate metabolism were investigated in hepatocellular carcinoma (HCC) induced in liver receptor homolog-1 (LRH-1) knockout mice. The influence of LRH-1 on glutaminase activity was monitored through the conversion of HP [5-13C]glutamine to [5-13C]glutamate, and the impact of LRH-1 deletion on the citric acid cycle could be investigated by conversion of HP [1-13C]pyruvate to four-carbon mitochondrial metabolites. The results demonstrate the potential of HP 13C MRS as a non-invasive method for understanding the role of oncogenic mutations in nuclear receptors in the metabolic reprogramming of cancer cells. Several other 13C-labeled substrates were studied, including [1-13C]cysteine and Î³-glutamyl[1-13C]glycine, a novel probe to detect gamma-glutamyl transferase (GGT) activity. The metabolic changes in T cells upon activation was investigated using HP [1-13C]cysteine as a potential selective probe of active T cells and HP Î³-glutamyl[1-13C]glycine to detect increased GGT expression by T cells in the active state. This work includes the first report on applications of HP 13C MRS in T cells and demonstrates the feasiblity to study the immune response with this technique. In conclusion, this work shows the potential of HP 13C MRS to investigate in vivo liver metabolism with administered 13C-labeled

[13C]bicarbonate labelled from hyperpolarized [1-13C]pyruvate is an in vivo marker of hepatic gluconeogenesis in fasted state

Josefina Adriana Maria Bastiaansen, Emine Can, Arnaud Comment, Rolf Gruetter, Hikari Ananda Infinity Yoshihara

Hyperpolarized [1-13C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by 13C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1-13C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by 13C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1-13C]pyruvate. The [13C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [13C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1-13C]pyruvate.

2022

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).

EPFL2012

Renal metabolism of hyperpolarized [1-13C]aspartate

Hikari Ananda Infinity Yoshihara

Aspartic acid is involved in several central metabolic pathways, including gluconeogenesis, the urea cycle, de novo nucleotide synthesis, the malate-aspartate shuttle, and via transmination the TCA cycle. The conversion in vivo of hyperpolarized [1-13C]pyruvate to aspartate, via pyruvate carboxylation to oxaloacetate, has been reported in rat kidney [1] and in the mouse liver [2]. Additionally, the metabolism of hyperpolarized [1-13C]aspartate to oxaloacetate has been noted in cultured cells supplemented with 2-oxoglutarate [3]. Aspartate is also of interest with its conversion to succinate in ischemic tissue implicated in subsequent reperfusion injury [4]. Here we report initial experiments with hyperpolarized [1-13C]aspartate in the rat kidney. [1-13C]Aspartic acid was formulated with OX063 trityl radical as in [3] as the tris salt, with the addition of glycerol (14% v/v) to improve glassing. The formulation is highly viscous and dissolves poorly, limiting the usable dose. Experiments were performed in a 9.4T animal scanner and a 7T polarizer operating at 1K, with automated rapid transfer and infusion. Rats (n=2) were isoflurane anesthesized, a femoral vein catheterized, and a quadrature 13C surface coil placed over the kidney. Respiration-triggered (TR ~3s) pulse-acquire (BIR4-30o) scans were started at the time of infusion of [1-13C]aspartate (~16 μmol/kg in 1.2 ml). Three metabolites were detected, with chemical shifts of 183.51 ppm (1.5%), 182.31 ppm (0.5%) and 181.26 ppm (0.6%), corresponding to [1-13C]malate, [4-13C]malate and an unidentified species provisionally assigned as N-acetyl[1-13C]aspartate. [4- 13C]aspartate was at natural abundance, but [4-13C]malate results from conversion to fumarate or succinate and label scrambling, and the greater prominence of [1-13C]malate indicates oxaloacetate as an intermediate. These results show the potential of hyperpolarized [1- 13C]aspartate as an in vivo metabolic probe.