Synopsis

In this study, the feasibility of studying neuronal energy metabolism in the mouse brain in vivo under [3-¹³C]-Lactate infusion, measured by ¹H-[¹³C] MRS at high magnetic field was evaluated. Brain tissue [3-¹³C]-Lactate enrichment and the turnover of its metabolic products were measured dynamically. Glutamate and glutamine C4 as well as glutamate+glutamine C3 were used as input to a one-compartment metabolic model of the neuronal TCA cycle and glutamate-glutamine cycle, enabling a comparison with previously reported values of metabolic fluxes in the mouse brain under infusion of other labeled substrates.

Introduction

Methods

All adult C57/BL6 (4 males and 2 females, 25.5±2.2g) were studied according to the local animal experimental guidelines and approved by the federal authorities.

MRS experiments were performed on a 14.1T magnet with a 26cm horizontal bore (Varian/Magnex) using a home-built ¹H surface coil in quadrature (two 9mm inner diameter loops) combined with a linear ¹³C coil (9mm diameter) as transceiver. In order to increase ¹³C measurement sensitivity, a localized proton-detecting carbon-editing sequence, i.e. BISEP-SPECIAL (1 and references therein) was used. B₀ homogeneity was optimized using first- and second-order shimming with FAST(EST)MAP (2), resulting in water linewidth of 25-30 Hz for a detection volume of 96µL (5.5×3.5×5 mm³). ¹³C-edited and non-edited spectra were acquired with 8-scan blocks in an interleaved mode (TE/TR=2.8/4000ms) during the entire infusion experiment.

[3-¹³C]lactate injection bolus dosage was calculated to reach 10% fraction enrichment (FE) based on the brain lactate level measured from the baseline acquisition without ¹³C-editing. A step-wise exponential decaying bolus was given in 15 minutes with five consecutive rate steps of 3 minutes and thereafter followed by a continuous administration of a [3-¹³C]-lactate solution (10% w/v 99% isotopic enrichment) mixed with 20% w/v unlabeled glucose and pH adjusted to 7. Thereafter, lactate infusion rate was continuously adjusted by visual estimation of the Lac C3 enrichment.

After frequency correction, every ten blocks (80 averages) of ¹³C-inverted and non-¹³C-inverted spectra were processed and quantified with LCModel (3), as previously described (1). Fractional enrichment (FE) was estimated from the resulting edited (¹³C) and non-edited (¹³C+¹²C) measurements, i.e. ¹³C/(¹³C+¹²C).

A one compartment model of brain energy metabolism was adapted from a previous study (1), as shown in Figure1. More specifically, through pyruvate dehydrogenase ¹³C label enters the tricarboxylic acid (TCA) cycle and labels C4 position of 2-oxoglutarate (OG) in the first TCA cycle turn. Via transmitochondrial transport OG exchanges with cytosolic glutamate (Glu) which gets labeled at position C4. In the second turn of TCA cycle, OG C3 receives the label from OG C4 and Glu C3 gets further labeled. Aspartate (Asp) gets labeled via the transmitochondrial exchange with TCA cycle intermediate oxaloacetate (OAA). C4 and C3 positions of glutamine (Gln) exchange ¹³C label with Glu C4 and C3 through Glu-Gln cycle. V_TCA, the TCA cycle rate; Vx, exchange flux between mitochondrial TCA cycle intermediates and cytosolic glutamate; V_NT, glutamate-glutamine exchange rate; dilN, the dilution factor of AcCoA pool induced by uptake of unlabeled plasma energy substrates.

Results and Discussion

The quality of the obtained spectra (Figure 2) enabled the visualization of cerebral LacC3 immediately after starting [3-¹³C]-lactate administration. Subsequently, alanine (Ala) C3 and glutamate (Glu) C4 appeared (Figure 3). Moreover, the turnover of Glu C4 and glutamine (Gln) C4, Glu+Gln C3 (GlxC3) and Glu+Gln C2 (GlxC2) could be resolved with a time resolution of 11min, during ~120 minutes of lactate infusion (Figure 3). After spectral quantification, a temporal resolution of 11min was obtained for FEs of LacC3, AlaC3 (Figure 4), GluC4, GlnC4 and GlxC3 (Figure 5). Using the measured LacC3 FE as input function, the one-compartment model was fitted to the measured GluC4, GlnC4 and GlxC3 turnover curves resulting in V_TCA= 0.59±0.07μmol/g/min, V_x= 1.97± 0.98μmol/g/min, V_NT= 0.23± 0.10μmol/g/min and dilN= 0.35±0.01.

We conclude that despite the low isotopic enrichment resulting from LacC3 uptake and metabolism in the mouse brain, localized in vivo ¹H-[¹³C]-MRS at 14.1T enabled a reliable measurement of the dynamic ¹³C labeling of amino acids and the determination of downstream brain metabolic rates consistent with previous glucose infusion studies (1). This opens the way to specific mice studies of blood lactate contribution as a fuel to brain energy metabolism.

Acknowledgements

References

1. Xin, L et al. Assessment of metabolic fluxes in the mouse brain in vivo using ¹H-[¹³C] NMR spectroscopy at 14.1 Tesla. Journal of cerebral blood flow and metabolism: 35, 759-765, doi:10.1038/jcbfm.2014.251 (2015)

2. Gruetter R, Tkac I. Field mapping without reference scan using asymmetric echo-planar techniques. Magn Reson Med 2000; 43(2): 319-23.

3. Provencher SW. Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med 1993; 30(6): 672-9.