Evaluation of Active Brown Adipose Tissue by the Use of Hyperpolarized [1-13C]Pyruvate MRI in Mice

Abstract

The capacity to increase energy expenditure makes brown adipose tissue (BAT) a putative target for treatment of metabolic diseases such as obesity. Presently, investigation of BAT in vivo is mainly performed by fluoro-d-glucose positron emission tomography (FDG PET)/CT. However, non-radioactive methods that add information on, for example, substrate metabolism are warranted. Thus, the aim of this study was to evaluate the potential of hyperpolarized [1-¹³C]pyruvate Magnetic Resonance Imaging (HP-MRI) to determine BAT activity in mice following chronic cold exposure. Cold (6 °C) and thermo-neutral (30 °C) acclimated mice were scanned with HP-MRI for assessment of the interscapular BAT (iBAT) activity. Comparable mice were scanned with the conventional method FDG PET/MRI. Finally, iBAT was evaluated for gene expression and protein levels of the specific thermogenic marker, uncoupling protein 1 (UCP1). Cold exposure increased the thermogenic capacity 3⁻4 fold (p < 0.05) as measured by UCP1 gene and protein analysis. Furthermore, cold exposure as compared with thermo-neutrality increased iBAT pyruvate metabolism by 5.5-fold determined by HP-MRI which is in good agreement with the 5-fold increment in FDG uptake (p < 0.05) measured by FDG PET/MRI. iBAT activity is detectable in mice using HP-MRI in which potential changes in intracellular metabolism may add useful information to the conventional FDG PET studies. HP-MRI may also be a promising radiation-free tool for repetitive BAT studies in humans.

Keywords: FDG PET; UCP1 expression; brown adipose tissue; cold exposure; hyperpolarized pyruvate MRI.

Figure 1

UCP1 protein and UCP1mRNA expression in interscapular brown adipose tissue (iBAT) biopsies. UCP1 protein levels were determined by Western blot (a representative Western blot is shown in Inset lower panel) and UCP1mRNA expression was determined by qPCR (Inset upper panel). For Western blot: n = 5 (cold) and n = 4 (thermo-neutral) and for qPCR: n = 5 (cold) and n = 4 (thermo-neutral).

Figure 2

Visualization of iBAT using hyperpolarized MRI (HP-MRI) and FDG PET-MRI. In (A) iBAT in a mouse under thermo-neutral and a mouse under cold conditions visualized by HP-MRI (transaxial projection) or FDG PET/MRI (transaxial and coronal projection) are shown (400× magnification). The solid arrow demonstrates the iBAT region and dashed arrows demonstrates the heart and large vessels (subclavian artery). The dotted arrow in the cold HP-MRI image represents a phantom. The color scale indicates [1-¹³C]signal intensity or FDG uptake using either HP-MRI or PET/MRI, respectively. The analysis was performed on spectra only from the regions of interest (ROI) drawn on the iBAT tissue. Due to slice thickness, signal from the heart and large vessels (subclavian artery) is seen in both thermo-neutral and cold animals. In (B) two representative iBAT [1-¹³C]spectra from a cold acclimatized (blue) and a thermo-neutral (red) mouse are shown. The [1-¹³C]signal is normalized to [1-¹³C]pyruvate in iBAT and frequency is measured in Hz.

Figure 3

Effect of cold versus thermo-neutrality on iBAT activity determined by HP-MRI. [1-¹³C]signal intensity of HP-MRI showing metabolites normalized to maximum [1-¹³C]pyruvate signal in iBAT during cold exposure and thermo-neutral conditions, respectively. In (A) the [1-¹³C]bicarbonate/[1-¹³C]pyruvate ratio is shown, (B) [1-¹³C]lactate/[1-¹³C]pyruvate ratio and (C) [1-¹³C]alanine/[1-¹³C]pyruvate ratio. n = 6 in each of the two groups. p-values are related to the comparison between thermo-neutral and cold group.

Figure 4

[1-¹³C]lactate/[1-¹³C]bicarbonate ratio corresponding to the ratio between anaerobic and aerobic metabolism in iBAT determined by HP-MRI. An increased [1-¹³C]lactate/[1-¹³C]bicarbonate ratio in the cold acclimatized mice by a 1.7-fold increment compared to thermo-neutral mice was observed, but this difference was not statistically significant (p = 0.429). n = 6 in each group.

Figure 5

The FDG PET/MRI and HP-MRI methods both imaging iBAT activity. The sum of [1-¹³C]-metabolites (∑[1-¹³C]-metabolites/ [1-¹³C]pyruvate) using the HP-MRI method is shown in (A). The signal of FDG PET/MRI is presented as percentage FDG activity in iBATcompared to whole body activity in a voxel of interest (VOI) marked (B). Cold exposure is compared to thermo-neutrality. For both methods; n = 3 in FDG PET/MRI study and n = 6 in the HP-MRI study.