We aimed to test if melatonin deficiency (due to surgical pinealectomy) decreased BAT activation, and if melatonin replacement could revert this pattern to normal in Wistar rats, using the gold standard for BAT detection in humans- 18F-FDG PET.
This is the first investigation, to our knowledge, to study BAT activation by 18F-FDG PET scan before and after melatonin treatment in animals. Despite data of increased BAT mass, UCP1 expression, and thermogenesis after melatonin replacement in many animal models and species already being demonstrated (3, 6, 14, 15), we found it relevant to investigate if 18F-FDG PET was able to detect these differences, facilitating future research in animals and humans, in which indirect measures of BAT are more feasible to be detected than through direct sample tissues. We believe that testing potential BAT activating compounds by performing an 18F-FDG PET in animals could help the translation of animal to human data. If animals increase their BAT SUV response to a specific compound, future studies can be done in humans to evaluate the same compound using the same method of detection.
In both isolated models (room temperature and after cold challenge), there were no differences between the groups, although some trends could be observed, including an unexpected higher maximal SUV in pinealectomized animals at room temperature and, on the contrary, a reduced maximal SUV in the P group after cold exposure. Since no statistical significance was achieved in any of the isolated methods, we prefer not to over interpret the reasons of this discrepancy. We should bear in mind that performing an 18F-FDG PET at room temperature is less sensitive to detecting differences; since the animals are chronically adapted to that temperature and have a small biological need to activate a recruited BAT. Perhaps in 23o C room temperature chronic exposure, which is not thermoneutral for rats, but clearly not as challenging as an acute 4o C exposure, other mechanisms of temperature regulation could be enough to maintain the normal thermoregulatory responses of the animal (3). Therefore, the absence of differences between the groups in this specific model was not unexpected, at first. There has been a lot of discussion about what the ideal temperature is to perform BAT and other metabolic studies in rodents, and this remains an open question (24, 25). The initial goal of performing the test at room temperature was exactly to observe if different patterns occurred when compared to the cold challenge and to calculate the ATC. A larger n could probably bring us more answers in future studies. As very few studies have analyzed BAT by PET in rats, our sample size calculation was based on a pilot study, and due to interindividual variances in Maximal SUV, we believe a larger sample size could have led us to results that were more conclusive.
However, comparing the actual ratio of BAT Maximal SUV increase, the acute thermogenic capacity (ATC), was also a primary goal, since it reveals if BAT is recruited to rapidly exert its effects on thermogenesis. In this context, the melatonin deficient group had a clearly reduced ATC, as expected. Since the PM and C groups had similar responses, this further confirms the hypothesis that melatonin proficiency is critical for thermoregulation in settings of acute cold challenging.
Indeed, the findings presented here encouraged our group to conduct an already published human study that demonstrated an increase in BAT volume and activity after melatonin replacement in a group of pinealectomized individuals (26). Interestingly, in this study, some individuals have a reasonably high baseline BAT activity before melatonin replacement; suggesting that even if melatonin is important for thermoregulation, other mechanisms still exist.
The UCP-1 RNA data in the present study was performed as an additional tool to analyze and interpret our image data. Importantly, other published manuscripts, some of them by our own group, have already shown a decrease in UCP-1 RNA and UCP-1 protein expression after pinealectomy and a reversal of this reduction by melatonin replacement in slightly different experimental models (5, 6, 14, 15). Therefore, regardless that our UCP-1 results are not original, they reinforce and validate the previous results and help in the interpretation of the image data (5, 6, 14, 15).
The observed pattern of UCP-1 expression in our model is in line with 18F-FDG PET results. The groups were statistically different; the P group has the lowest UCP-1 expression, compared with the other two groups, which in some ways could explain the lower ATC.
However, as already pointed out, an increase in UCP-1 RNA does not necessarily mean more heat production, as this recruited tissue can be inactive, if there is no need to increase thermogenesis (3, 16, 17). Even differences in RNA and protein expression could arise, as post-transcriptional factors may influence protein synthesis. This could explain why the pinealectomized group showed a normal BAT response in 18F-FDG PET at room temperature, even with having lower UCP-1 expression. We did not expect differences in UCP1 expression between the C and PM groups, since our intent was to reproduce, immediately after pinealectomy, in the PM group, the physiological production of melatonin. Nevertheless, clear differences were observed, so the main hypothesis is that the replacement protocol did not exactly mimic melatonin production. As the animals drink water at specific times of the night, they would probably have several small melatonin peaks, instead of the physiological curve of night melatonin production. In addition, since the melatonin solution was only available from the start of the dark period, the duration of its plasmatic profile might reflect the winter type of melatonin secretion in some animals, changing the physiological pattern of BAT recruitment. These different kinetic curves could explain the differences observed in figure 4.
Our main focus in this particular study was to evaluate BAT responses in 18F-FDG PET. As many other different studies have demonstratedthe physiological role of melatonin in body weight regulation, food intake, energy expenditure, metabolic risk factors, and many other parameters, we did not include these data as they would be redundant. Our group has previously shown that pinealectomy leads to a metabolic syndrome phenotype in rats and melatonin replacement reverts it (1). Melatonin has been shown to decrease body weight with a minimal decrease in food intake, suggesting an effect in energy expenditure, proposed to be mediated by BAT (2, 3). For a more comprehensive review of metabolic consequences of melatonin deficiency and the physiological role of melatonin in several animal models, see references 1 and 2.
The research presented here aggregate data on the role of melatonin on thermoregulation, but the model has several limitations. Many questions still persist. Is there a role of melatonin in maintaining thermoneutrality in animals at room temperature, or is it only important if the temperature acutely decreases? Would the same results appear if the cold challenge was less intense and had a longer duration, probably a more physiological challenge? Would ATC be a reliable way to detect recruited BAT in vivo, as our study suggests? We believe so, but to detect clear effects of different compounds, we need to compare differences between the room temperature and cold challenge, since many adaptive mechanisms should exist, as thermoneutrality is vital for survival. Performing exams in only one of these conditions could lead to misinterpretations of the role of BAT and possible recruiters on thermogenesis and there is clear evidence that small differences in laboratory temperatures could lead to very different results in different animal models (24, 25, 27-29). For example, if the tests were performed only after the cold challenge, we could conclude that no differences were seen between the groups, despite large differences in UCP-1mRNA expression. This could have led to an interpretation that UCP-1 is expressed but not necessarily translated, or that FDG-PET is not a reliable way to detect differences in BAT activation in animals.
BAT physiology is very complex, and potential compounds aiming to increase BAT can act in recruitment, activation, or both (3, 7, 11, 12). Experimental models can help us distinguish between the action of potential BAT recruiters, such as melatonin, and the detection of BAT in vivo, by imaging techniques. Different models have the potential to investigate the role of several recruiters in different laboratory conditions, paving the way for human studies with potential compounds capable of activating or recruiting BAT, besides melatonin (11, 30). Our finding that melatonin seems critical for acute BAT activation, induced by cold and measured by 18F-FDG PET, is novel. It can lead to future imaging studies in both animals and humans that will help understand the physiological role of melatonin, concerning BAT, and if melatonin could be a potential target for increasing melatonin recruitment and activation in humans, with potential therapeutic use in metabolic diseases (26). In the same way, the finding of reduced melatonin production, leading to reduced BAT thermogenic responses, can help the understanding of increased light-at-night exposure as a potential risk factor for obesity and metabolic diseases, as already suggested (31, 32).