BAT is a fat tissue that regulates energy balance and maintains core body temperature through sympathetic NST(1, 35). Insufficient BAT activity could lead to an energy imbalance, resulting in metabolic diseases like obesity and diabetes. As such, BAT is a promising target for therapies and treatments for obesity(6). Detection and quantification of BAT thermogenesis in humans is commonly done by using 18F-FDG PET/CT imaging after mild cold exposure(23). However, to assess differences in BAT thermogenic activity across subjects, or to monitor changes in BAT thermogenesis in the same subject, 18F-FDG uptake ought to reliably reflect the degree of thermogenic activity in BAT. The aim of this study was to assess whether there exist differences in 18F-FDG uptake between animals with very different functional BAT thermogenic capacity, while addressing some of the inconsistencies found in the literature regarding varying 18F-FDG uptake patterns for mice with functional or impaired BAT(16–19).
Since animal handling in awake mice is known to lead to a considerable and variable stress response, resulting in the release of hormones and glucocorticoids that may influence the degree of BAT activity, all experiments were done in anesthetized mice. For these studies, all mice were anesthetized with pentobarbital, one of the few anesthetics that do not adversely affect BAT thermogenic capacity(21, 22).
In response to cold stress, NE is secreted and attaches to β3-adrenergic receptors on brown adipocytes to initiate the signal pathway for BAT thermogenesis. This provides UCP1-carrying mice the means to survive cold environments. But, UCP1-lacking mice are also capable of adapting to colder conditions by developing endurance for muscular shivering. Therefore, the participation of BAT in response to cold-stress is completely optional(1). In fact, since all mice were kept in a chronic mild cold environment at 24 °C, this could influence the baseline values observed for WT and KO mice. In order to isolate the effect of BAT thermogenesis on glucose uptake in BAT, we decided to inject NE directly into the anesthetized mice, bypassing any uncontrollable adaptive response to cold-exposure that KO mice might have developed.
In preparation for 18F-FDG PET scans, it is common practice to have subjects fast. This is because elevated blood glucose levels have variable effect on SUV measured within different organs. For example, SUV in the brain and liver may decrease due to the endogenous glucose competitively inhibiting the uptake of the exogenous, glucose-analog radiotracer(36). On the other hand, a previous study had shown a remarkable decrease in BAT uptake in non-anesthetized mice upon fasting(37). For our study, experiments were run on two different sets of mice. Set 1 consisted of mixed-sex WT and KO mice that were scanned unfasted. Set 2 consisted of all male WT and KO mice that were scanned fasted. Interestingly, we did not see any significant difference in 18F-FDG uptake between fasted and unfasted mice (p = 0.3 for KO, and p = 0.9 for WT). This should not be surprising, though, as the reduction in glucose uptake seen in awake, fasted mice(37) is more likely due to the reduction in the sympathetic nervous system response of brown adipose tissue(38), which is clearly not seen in our studies in which BAT was directly stimulated by NE. Interestingly and somewhat counter intuitively, fasting led to an even higher standard deviation of glucose uptake in BAT in both WT and KO mice. More insight could be gained in a future study by measuring individual glucose tolerance in order to normalize glucose uptake.
Figure 7 shows how NE injection affects SUVpeak. For WT mice, SUVpeak increased significantly with NE stimulation of BAT, whereas there was no consistent change in 18F-FDG uptake in KO mice. At baseline, higher 18F-FDG uptake was seen in KO mice. It is important to acknowledge the fact that our baseline scans were collected one week after the NE treatment scans. It has been shown that thermogenic activation can affect the functionality of BAT of KO mice through inflammation or other pathways(39, 40). Our study would have benefitted from collecting baseline data before the treatment data, and preferably on the same day. Thus, it is difficult to draw conclusions based on the comparison of means.
On the other hand, a comparison of the variances is much more interesting. At baseline, the variance for KO mice was much greater than WT mice (p < 0.001). But, with the inclusion of the NE treatment, the SUVpeak variances between KO and WT were comparable (p = 0.9). We hypothesize that this variability seems to suggest that, for WT mice, there is some UCP1-dependent mechanism that inhibits glucose uptake in absence of adrenergic stimulation, while facilitating glucose uptake upon stimulation. Glucose transport across cell membranes is facilitated by proteins such as glucose transporter 1 (GLUT1). Inokuma et al.(17) reported that GLUT1 mRNA levels were 1.4 times higher in KO mice when compared to WT mice. Therefore, even without stimulation, KO mice are expected to have higher 18F-FDG uptake due to higher expression of GLUT1. The functional activity of GLUT1 is enhanced after NE injection, and this may be due to a conformational change in GLUT1 that increases its affinity for glucose(41). But, as shown in Fig. 5, this conformational change might be UCP1-dependent. In the presence of UCP1, GLUT1 might behave like a NE-gated channel that either restricts glucose uptake at baseline, or enhances glucose uptake upon NE stimulation. Conversely, for KO mice that lack UCP1, such control on GLUT1 might have lost, resulting in consistent changes and high variations in both baseline and stimulated conditions.
For Set 1, a wider range of SUVpeak was observed in female compared to male mice. In addition, a significant overlap of SUVpeak was seen between WT and KO female mice (Fig. 2). These findings are somewhat consistent with those observed by Jeanguillaume et al.(18) and Hankir et al.(19). The wider range in SUVpeak observed in female mice could be ascribed to differences in estrogen levels, possibly present as mice were not scanned at the same estrous cycle time point (24). As the resources required to monitor blood estrogen level were not available at our facility, to limit possible variations in SUVpeak due to differences in estrogen level, female mice were excluded from Set 2 for the second study. Future studies should explore the effect of estrogen levels, that could be measured with various kits for serum measurement(42) or urine samples(43), on glucose 18F-FDG uptake.
Differences in BAT thermogenic capacity between WT and UCP1 KO mice are well established in the literature. In our study, the lack of UCP1 in the BAT of KO mice and the presence of UCP1 in BAT of WT mice was established by genotyping of mouse tail DNA via PCR, as well as by immunohistochemistry staining of excised BAT. Differences in BAT thermogenic capacity between the two phenotypes were also assessed by rectal temperature measurement and by using infrared thermography. While rectal temperature measurements showed a much higher core body temperature increase in WT mice than KO mice 40 minutes after NE injection, further demonstrating different NE thermogenic response between WT and KO mice, infrared thermography did not show any statistically significant difference in thermogenic capacity between WT and KO mice, consistently with previous studies done in unconscious mice showing the inability of thermal imaging to show changes with CL-316,243(44). As such, in our study, while infrared thermometry was able to detect body temperature increase in both animals following adrenergic stimulation, it was not able to detect differences in BAT thermogenic activity between WT and KO mice, which is consistent with previous findings(34).