Detection of brown adipose tissue in rats with acute cold stimulation using quantitative susceptibility mapping : Chinese Medical Journal

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Detection of brown adipose tissue in rats with acute cold stimulation using quantitative susceptibility mapping

Zhu, Cuiling1,2; Guo, Yihao3,4,5; Si, Wenbin3; Zhong, Qiaoling1; Mei, Yingjie6; Feng, Yanqiu3,4,5; Zhang, Xiaodong1

Editor(s): Guo, Lishao

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Chinese Medical Journal: November 07, 2022 - Volume - Issue - 10.1097/CM9.0000000000002388
doi: 10.1097/CM9.0000000000002388

To the Editor: Adipose tissue occurs in at least two different entities in mammals and humans: brown adipose tissue (BAT) and white adipose tissue (WAT). BAT is characterized by a unique uncoupling protein 1 (UCP1) in the mitochondria that enables the uncoupling of the respiratory chain from adenosine triphosphate synthesis. Thus, energy is dissipated as heat to reduce fat accumulation. BAT is also considered a highly heterogeneous tissue with abundant oxygen, blood supply, and iron-rich mitochondria.[1,2] Activation of BAT via exposure to a cold environment is considered to be a means of reducing triglycerides to fight obesity.[3] The alterations in cells and tissues of activated BAT include increased iron content and UCP1 expression in mitochondrial, blood perfusion, and lipid utilization.[4] Therefore, accurate identification and quantitative analysis of inactive and activated BAT are of great significance for the treatment of metabolic diseases that target BAT, such as obesity.

Owing to the iron-rich mitochondria in BAT,[5] we tested our hypothesis that quantitative susceptibility mapping (QSM) could be a promising method for monitoring BAT activation in the interscapular region and showing significant differences in magnetic susceptibility in WAT from BAT. In the present study, we aimed to investigate the feasibility of QSM for differentiating BAT from WAT and detecting activated BAT in the interscapular region of rats, with comparisons to fat fraction (FF) and R2*.

All animal experiments were conducted in accordance with the Southern Medical University Animal Care and Use Committee guidelines. Sprague–Dawley male rats (5 weeks old, 150–200 g) were acclimated to our animal facility maintained at 22°C with 12 h light and dark cycles for one week. The rats were subsequently divided into three groups: control group (Control), cold-stimulated group for 12 h (Cold 12 h), and cold-stimulated group for 24 h (Cold 24 h). The rats in the control group were exposed to room temperature. Those in the Cold 12 h and Cold 24 h groups were exposed to cold at 4°C for 12 h and 24 h, respectively. After the cold stimulation, a three-dimensional multi-echo gradient-echo sequence was performed on a 7T MR system (Bruker, Bremen, Germany). The animals were placed in the coil headfirst in the supine position for the interscapular region scanning. All animals were sacrificed immediately after magnetic resonance imaging (MRI) experiments. BAT and WAT depots were excised from the scanning region for histological analysis and the special protein expression quantification. The experimental design is shown in Supplementary Figure 1 ( The MRI scan parameters and data analysis are shown in the Supplementary Materials (

MRI maps highlight the unique wings shape of BAT and a triangular sheet of WAT [Figure 1A]. Hematoxylin-eosin staining showed the number of lipid vacuoles decreased in the activated BAT with cold stimulation for 12 and 24 h. In addition, high-density cells with plenty of cytoplasm were observed in BAT after cold stimulation. However, no obvious change in lipid vacuoles was observed in WAT after cold stimulation [Figure 1B]. By contrast, the small vessels in WAT were dilatated and proliferated after cold stimulation. Immunohistochemical staining showed a large amount of positive expression of UCP1 in BAT, which indicates that BAT was activated effectively, but without positive expression in WAT [Figure 1B]. Western blotting analysis revealed that UCP1 of BAT expressed continuously in the three groups, and the expressions were more statistically significantly upregulated in both cold 12 h and 24 h than those in the control group [Figure 1D].

Figure 1:
(A) Axial slices of the reconstructed FF, QSM, and
maps of interscapular BAT and WAT in the different groups. FF, QSM, and
maps of a normal male rat without cold exposure (top), cold stimulated male rats with 12 h (middle) and 24 h (bottom). White long and short arrows indicated the contour of BAT and WAT, respectively. (B) Representative histology of H&E and immunohistochemistry of UCP1 in BAT (left) and WAT (right) (original magnification × 100). (C) Column diagram analysis of FF, QSM, and
between WAT and BAT in three groups (Control, Cold 12 h, and Cold 24 h). Column diagram analysis of FF, QSM, and
of BAT or WAT in different groups. P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. (D) Western blotting showed that UCP1 expressions at 4°C exposed in interscapular BAT were statistically significantly higher than those of the control group. Interscapular BAT UCP1 expression increased in sequence in the groups of Control, Cold 12 h, and Cold 24 h. BAT: Brown adipose tissue; FF: Fat fraction; H&E: Hematoxylin-eosin; QSM: Quantitative susceptibility mapping; UCP1: Unique uncoupling protein 1; WAT: White adipose tissue.

Our results showed significant differences in QSM, FF, and R2* between BAT and WAT whether in room temperature (inactive) or cold exposure (activated) (P < 0.05). In line with previous studies,[6,7] FF decreased significantly in inactive or activated BAT compared with WAT. Our study additionally found that BAT had a statistically significantly lower QSM in contrast to WAT. Therefore, the QSM value can effectively identify BAT from WAT under room temperature or cold exposure.

By contrast, QSM, FF, and R2* of cold-stimulation activated BAT (Cold 12 h or 24 h groups) were all significantly lower than inactive BAT (control group), and QSM was further significantly reduced after cold stimulation for 12 h or 24 h [Figure 1C]. The change in QSM value could be attributable to the comprehensive influence of iron content in mitochondria and fat content of BAT after acute cold stimulation. The increasing iron content and the decreasing fat content have opposite effects on the change of susceptibility because they are all paramagnetic substances. If the increasing iron content is not enough to compensate for the decreasing fat content in tissue, the total susceptibility will decrease. This final analysis indicated that the change of fat content in BAT after cold stimulation could play a dominant role in total magnetic susceptibility rather than the iron content in mitochondria. Histologically, the number of adipose vacuoles was confirmed to decrease significantly in BAT under cold exposure. As a consequence, the QSM value can distinguish between activated and inactive BAT.

We also found that the cold stimulation groups, regardless of 12 h or 24 h, induced a significantly decreased QSM in WAT compared with the control group (0.19 ± 0.12 ppm vs. 0.55 ± 0.09 ppm, 0.21 ± 0.13 ppm vs. 0.55 ± 0.09 ppm), while no significant changes of FF and R2* were observed. We speculated that the change of QSM in WAT might be related to the dilatation or proliferation of small vessels in WAT after cold stimulation. Further pathologic or histologic validation may prove QSM to be useful in the detection of tiny change of cell structure while FF and R2* do not reach the detection in WAT.

A best linear relationship between FF and UCP1 expression was found (r = −0.7930), followed by QSM, suggesting that FF may correlate well with the activated state of BAT with consistent to the previous study [Supplementary Figure 2,].[8] To our knowledge, magnetic susceptibility is not only affected by the changes of iron in mitochondria but also the content of fat in BAT. Although FF has a higher correlation with UCP1 expression than QSM and R2*, the limitation is the observed change of FF only based on fat utilization rather than additional BAT microstructure. Using FF as an absolute or single indicator to detect activated BAT from inactive BAT may not be accurate. The values of QSM and FF together may be a better choice to identify activated BAT from inactive BAT.

In conclusion, our study found that the inactive or activated BAT has a lower QSM and FF and a higher R2* compared with WAT. Additionally, the activated BAT induced by acute cold stimulation has lower QSM, FF, and R2* than inactive BAT. All the MRI biomarkers mentioned above are confirmed with a good linear correlation with UCP1 expression of BAT. To our knowledge, QSM is highly sensitive for iron and fat content detection and susceptibility contrast directly reflects subtle variations in tissue composition consistent with histological changes. QSM provides more details about the identification of substructures of BAT and WAT, compared with the other MRI biomarkers such as FF and R2*. QSM could be a new quantitative biomarker for identifying and detecting BAT.


This study was supported by a grant from the National Natural Science Foundation of China (No. 81801653).

Conflicts of interest



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