In vivo metabolic studies in skeletal and cardiac muscle using ¹³C magnetic resonance spectroscopy

Cardiac and skeletal muscle function relies on a continuous energy production via fatty acid metabolism and mitochondrial oxidation of pyruvate, with a fine balance between substrate delivery and utilization. Changes in metabolism are increasingly being implicated as playing an intrinsic role in many diseases, such as diabetes, cancer, and heart failure. Investigating and identifying fundamental metabolic processes are paramount to understanding pathologies. Beyond morphology and functional information, magnetic resonance can provide insights at a metabolic level using spectroscopic techniques such as 13C NMR. The low natural abundance and sensitivity of the 13C nucleus makes 13C NMR in biological systems challenging. In addressing this issue, hyperpolarized methods have emerged as a very promising tool, obtaining signal enhancements up to 10,000 fold. Spectra of hyperpolarized 13C labeled substrates and their downstream metabolic products offer insight into metabolic processes occurring in vivo within seconds after the injection. This thesis focused on the development of MR hyperpolarization methods and applications to study energy metabolism in cardiac and skeletal muscle in vivo. This ranged from development of the experimental frame work to mathematical tools to characterize the observed metabolic processes. Methods were developed to visualize 13C labeling kinetics of acetylcarnitine in vivo in resting skeletal muscle following the administration of hyperpolarized [1-13C]acetate. Two different, novel mathematical models were constructed to quantify the kinetic rate constants. Although separated by two enzymatic reactions, the conversion of acetate to acetylcarnitine was uniquely defined by the enzymatic activity of acetylCoA synthetase (ACS). A 13C MRS protocol was developed and implemented for hyperpolarized studies in the heart, which included selecting appropriate cardiac triggers to align the measurements with the cardiac phase. The 13C label propagation into acetylcarnitine and citrate could be measured in real time in the beating rat heart following the infusion of hyperpolarized [1-13C]acetate, using a newly constructed 13C RF coil which improved the detection sensitivity. The substantial spectral resolution at 9.4T and a triggered shimming and MR acquisition protocol allowed for the detection of citrate for the first time in vivo after injection of hyperpolarized [1-13C]acetate. Mathematical models were successfully extended to include mitochondrial oxidation and analytical expressions were derived to interpret the dynamic 13C labeling of citrate. Cardiac dysfunction is often associated with a shift in substrate preference, while diagnostic methods such as PET provide only information on substrate uptake. The potential of hyperpolarized 13C MRS to measure simultaneously lipid and carbohydrate oxidation was demonstrated in vivo, and the sensitivity of the method to a metabolic perturbation was assessed. Hyperpolarized [1-13C]butyrate and [1-13C]pyruvate were used as representatives of carbohydrate and lipid oxidation. Fasting led to significant changes in preference for the injected substrates. The appearance of a cohort of downstream metabolites (bicarbonate, lactate, alanine, glutamate, citrate, acetylcarnitine, β-hydroxybutyrate and acetoacetate) allowed the independent and simultaneous monitoring of myocardial oxidation of both fatty acid and carbohydrates in vivo and is a sensitive indicator of metabolic shift. Lactate is an important metabolic intermediate for mitochondrial oxidation. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1-13C]lactate, which can be injected at physiological concentrations and leaves other oxidative processes undisturbed. A significant decrease in [1-13C]alanine and 13C bicarbonate were observed comparing both groups, attributed to a change in cellular alanine concentration and pyruvate dehydrogenase (PDH) flux. It was shown that lactate can be used to study carbohydrate oxidation in skeletal muscle at physiological levels and that the downstream metabolite signals are sensitive markers to probe metabolic changes. Since the detection of [5-13C]citrate and [5-13C]glutamate in the heart is hindered by the close proximity of the [1-13C]acetate resonance, acetylcarnitine could be an interesting substrate for hyperpolarized MR. Acetylcarnitine crosses the mitochondrial membrane easily, while skipping a few metabolic steps needed for acetate to cross the membrane. Moreover, it does not interfere with the detection of [5-13C]glutamate and [5-13C]citrate. [1-13C]Acetylcarnitine was successfully hyperpolarized and despite its short T1 it was possible to detect the formation of [5-13C]glutamate in the heart in vivo. Due to the absence of the [5-13C]citrate resonance, we hypothesized the existence of an intricate relationship between reaction, transport and relaxation rates in the choice of hyperpolarized substrates. Acetylcarnitine has relatively small poolsizes and has only been observed using hyperpolarized 13C MRS techniques. Therefore, it has not been possible to obtain reliable quantification of the metabolic turnover of acetylcarnitine, which is an essential intermediate in the metabolism of acetate. Methods were implemented to measure the oxidation of [2-13C]acetate locally with increased sensitivity at high field (14.1T), using a 1H-13C polarization transfer sequence. This allowed the observation of [2-13C]acetylcarnitine in vivo at 21.5 ppm, with a metabolic turnover time τ of 0.34 μmol/g/hr. The acetylcarnitine resonance assignment was confirmed by experiments infusing 13C labeled glucose. The measurement of the time courses of Glu C4 and C3 were also clearly observed, without lipid contamination. To conclude, this work presents novel metabolic information about skeletal and cardiac energy metabolism using developed methods in hyperpolarized 13C MRS. The use of hyperpolarized 13C substrates to measure absolute flux through metabolic pathways in vivo would not only revolutionize clinical diagnostic imaging but also serve as an important tool to better understand diseased metabolism and the metabolic effects of new drugs aimed to combat diseases.

Hyperpolarized 13C lactate as a substrate for in vivo metabolic studies in skeletal muscle

Josefina Adriana Maria Bastiaansen, Arnaud Comment, Rolf Gruetter, Yuhei Takado, Hikari Ananda Infinity Yoshihara

Resting skeletal muscle has a preference for the oxidation of lipids compared to carbohydrates and a shift towards carbohydrate oxidation is observed with increasing exercise. Lactate is not only an end product in skeletal muscle but also an important metabolic intermediate for mitochondrial oxidation. Recent advances in hyperpolarized MRS allow the measurement of substrate metabolism in vivo in real time. The aim of this study was to investigate the use of hyperpolarized 13C lactate as a substrate for metabolic studies in skeletal muscle in vivo. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1-13C]lactate, a substrate that can be injected at physiological concentrations and leaves other oxidative processes undisturbed. 13C label incorporation from lactate into bicarbonate in fed animals was observed within seconds but was absent after an overnight fast, representing inhibition of the metabolic flux through pyruvate dehydrogenase (PDH). A significant decrease in 13C labeling of alanine was observed comparing the fed and fasted group, and was attributed to a change in cellular alanine concentration and not a decrease in enzymatic flux through alanine transaminase. We conclude that hyperpolarized [1-13C]lactate can be used to study carbohydrate oxidation in resting skeletal muscle at physiological levels. The herein proposed method allows probing simultaneously both PDH activity and variations in alanine tissue concentration, which are associated with metabolic dysfunctions. A simple alteration of the nutritional state demonstrated that the observed pyruvate, alanine, and bicarbonate signals are indeed sensitive markers to probe metabolic changes in vivo.

2014

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

Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-(13)C]butyrate and [1-(13)C]pyruvate

Josefina Adriana Maria Bastiaansen, Arnaud Comment

Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) (13)C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-(13)C]pyruvate and [1-(13)C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [(13)C]bicarbonate (-48%), [1-(13)C]acetylcarnitine (+113%), and [5-(13)C]glutamate (-63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-(13)C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-(13)C]acetoacetate and [1-(13)C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-(13)C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (-82%). Combining HP (13)C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease.

2016

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

Bernard Lanz

EPFL2012