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Unveiling the fate of glycolytic substrates using multi-spectral CEST: proof of concept with 2DG in the rat brain
Yohann Mathieu-Daudé1,2, Mélissa Vincent1,2, Julien Valette1,2, and Julien Flament1,2
1Molecular Imaging Research Center (MIRCen), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), Fontenay-aux-Roses, France, 2UMR 9199, Neurodegenerative Diseases Laboratory, Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, Université Paris-Saclay, Fontenay-aux-Roses, France

Synopsis

2-Deoxy-D-glucose (2DG), an analogue of glucose similarly transported but with metabolism blocked after the first phosphorylation into 2DG-6-phosphate (2DG6P), has already been used to study glycolytic metabolism using gluCEST. However, origin of glucoCEST signal is still an open question important to be addressed. In this study, we measured for the first time variations of CEST signal in the rat brain at different resonance frequencies following 2DG injection. The richness of CEST signal can help assessing the fate of glycolytic substrates and would constitute a first step toward quantitative measurement of glucose metabolism using CEST method.

Introduction

Glucose degradation contributes to several biochemical reactions that provide energy to cells. Recently, glucoCEST1,2 has been proposed as an alternative to 18F-FDG to study glucose consumption using injection of glucose or 2-deoxy-D-glucose (2DG), an analogue similarly transported but with metabolism blocked after the first phosphorylation into 2DG-6-phosphate (2DG6P)2-5. While glucose and 2DG exhibited comparable glucoCEST effects in phantoms, intravenous injection of 2DG has been reported to lead to higher glucoCEST signal accumulation, probably because 2DG6P remained trapped for longer time into the cells2,3. GlucoCEST signal may potentially arises from 3 different compartments i.e. vascular, extravascular/extracellular and intracellular (Fig.1). Even if such data argue in favor of intracellular origin of CEST contrast, some other works reported primarily extracellular origin6 or both1,5. Thus, compartmental origin of glucoCEST signal is still an open question important to be addressed for further utilization of glucoCEST. Surprisingly, in spite of the very complex form of the MTRasym curves of glucose or analogues, which exhibit 4 different peaks related to non-equivalent exchanging hydroxyl groups (Fig.2), all studies only reported variations of CEST signals measured at one saturation offset (~+1 ppm)7-9. Here, we measured for the first time variations of CEST signals at 4 different offsets following 2DG injection in rats, possibly providing deeper insights into the origin of glucoCEST signal.

Material & Methods

GlucoCEST sequence: To maximize detection sensitivity over spatial resolution, we used a spectroscopic approach based on fully adiabatic LASER localization preceded by a CEST module consisting in a frequency-selective continuous wave pulse (Tsat = 4s, B1 = 2µT). Acquisitions were performed on a horizontal 11.7T Bruker magnet using volume coil in transmission (homogenous B1 field) and a quadrature surface coil in reception.
Phantom acquisitions: Solutions of 50mM of 2DG and glucose-6-phosphate (G6P) were prepared. B1 intensity was ranged between 1.2 and 4.0µT with a 0.4µT step and saturation offsets were ranged between -5 to 5ppm with a 0.2ppm step.
Animal acquisitions: 4 rats under isoflurane anesthesia (1.5%) were injected intravenously with a 2DG solution (20% w/w) in 8 minutes for a total dose of 2DG of 1g/kg. A voxel of 2.5x2.5x2.5mm3 was placed in left striatum and glucoCEST spectrum were acquired in 1.5min with B1 = 2µT and saturation offsets applied at -20, ± 2.9, ± 2.1, ± 1.2 and ± 0.8ppm. Zspectra were acquired during 12.5min before 2DG injection and 122.5min after.

Results

MTRasym curves acquired in 2DG and G6P phantoms showed very different profiles (Fig.2.a and b respectively). 2DG exhibited 4 well resolved peaks corresponding to 3 different hydroxyl groups (peaks at 2.1 and 2.9ppm corresponding to α- and β- anomeric forms of the pyranose) while G6D exhibited broader peaks. Interestingly, strongest CEST signals were measured at 0.8 and 1.2ppm for 2DG (Fig.2.c) whereas maximal CEST signals were measured at 2.1 and 2.9ppm in G6P (Fig.2.d). While no variation was observed at 0.8ppm, probably due to the poor sensivity of the method close to water resonance frequency (Fig.3.a), increased glucoCEST contrast was measured at 1.2, 2.1 and 2.9ppm after 2DG injection in rats (Fig.3.b, c and d respectively, black dotted line) followed by a return to baseline level. To better visualize differences between offsets, curves were normalized and kinetics were compared (Fig.4). While glucoCEST uptakes measured at 2.1 and 2.9ppm exhibited similar timing (Fig.4.a), they were delayed compared to 1.2ppm (Fig.4.b). Moreover, plateau durations, increase and decrease times were also different between 1.2ppm and 2.1/2.9ppm (Fig.5).

Discussion and conclusion

Thanks to very different MTRasym profiles, it is possible to discriminate 2DG and G6P. Note that G6P and not 2DG6P was used for phantom acquisition. Nonetheless, the missing hydroxyl group on 2DG6P (compared to G6P) resonates at 1.2ppm10,11 so it would not affect CEST signal at 2.1 and 2.9ppm measured in G6P phantom.
Interestingly, CEST signals at 2.1 and 2.9ppm were 2.5min delayed compared to 1.2ppm and times to return to baseline were also longer (trb, Fig.5). If we consider that CEST signal is mostly 2DG6P-weighted at 2.1 and 2.9ppm, it is consistent with 2DG6P accumulation in the intracellular space produced by 2DG phosphorylation by hexokinase. This is coherent with previous works reporting that early data points after injection mostly reflect 2DG transport in extravascular/extracellular and intracellular spaces whereas later time points reflect 2DG6P accumulation in cells2,5. Surprisingly, decrease of glucoCEST contrast observed after ~60min would suggest 2DG and 2DG6P degradation or washout. Similar effect has already been reported2 and could correspond to degradation of 2DG6P through pentose phosphate pathway2 glycogen conversion12,13, further metabolism in 2DG-1-P and 2DG-1,6-P14 or passive washout due to osmotic pressure. Finally, we reported for the first time an undershoot of glucoCEST signal at 2.1 and 2.9ppm before returning to baseline (Fig.3.c, d and 4.a). Such an effect, which was not observed after glucose injection (data not shown), is still under investigation. It could be due to modification of intracellular glucose concentration in response to high dose of 2DG. Thanks to resolving CEST variation at different resonance frequencies, it seems that 2DG uptake, consumption and degradation by cells can be assessed with unprecedented richness. This would constitute a first step toward quantitative measurement of glucose metabolism using CEST.

Acknowledgements

This work was supported by a grant from Agence Nationale pour la Recherche (“nrjCEST” project, ANR-18-CE19-0014-01). The 11.7 T scanner was funded by a grant from NeurATRIS: A Translational Research Infrastructure for Biotherapies in Neurosciences (“Investissements d'Avenir”, ANR-11-INBS-0011).

References

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Figures

Figure 1: Compartmental origin of glucoCEST signal. After injection into the intravascular space, 2DG enters extravascular/extracellular space before being internalized by cells. It is then phosphorylated into 2DG6P by hexokinase. If further metabolism of 2DG6P through glycolysis is blocked, others pathways may explain 2DG6P degradation.

Figure 2: In vitro characterization of 2DG and G6P CEST effects. MTRasym were acquired in 50mM solution of 2DG (a) and G6P (b) with peaks attributed to each hydroxyl groups. Evolutions of MTRasym in function of B1 intensity exhibited very different profiles at each saturation offset in 2DG (c) and G6P (d).

Figure 3: In vivo kinetics of glucoCEST signal. Intravenous injection of 2DG (1g/kg) was performed in 4 rats and glucoCEST variations were measured before and after 2DG injection (black dotted line) at 0.8ppm (a), 1.2ppm (b), 2.1ppm (c) and 2.9ppm (d). Mean variations of CEST signal compared to baseline were shown (n = 4, mean±SEM).

Figure 4: Comparison of CEST signal variation timing. Variation of CEST signals acquired at 1.2, 2.1 and 2.9ppm were scaled to compare their relative timings. While signals at 2.1 and 2.9ppm showed similar timing (a), they were delayed compared to 1.2ppm (b).

Figure 5: Quantification of kinetics parameters. Kinetics parameters were quantified for CEST variations measured at 1.2, 2.1 and 2.9ppm. Shift: delay between 2DG injection and CEST signal increase; d1 and h1: rise time and increase of CEST signal; pd: plateau duration; d2 and h2: fall time and decrease of CEST signal; trb: time between 2DG injection and return to baseline of CEST signal.

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)
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