Melatonin increases brown adipose tissue acute thermogenic capacity in rats measured by 18F-FDG PET

Objective Melatonin has been shown to increase brown adipose tissue (BAT) mass, which can lead to important metabolic effects, as bodyweight reduction and glycemic improvement. However, BAT mass can only be measured invasiveness, and the gold-standard for non-invasive measurement of BAT activity is positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro-D-glucose ( 18 F-FDG PET). There is no study, to our knowledge that evaluated if melatonin influences BAT activity measured by this imaging technique. Methods Three experimental groups (control, pinealectomy, and pinealectomy replaced ith melatonin) performed 18 F-FDG PET in ambient temperature and after acute cold exposure. The ratio of increased BAT activity after cold exposure/ambient temperature was called “acute thermogenic capacity.” We also measured UCP-1 mRNA expression to correlate with 18 F-FDG PET results. Results Pinealectomy led to a reduced acute thermogenic capacity compared with the other groups, as well as a reduced UCP1 mRNA expression. Conclusion Melatonin deficiency apparently impairs BAT response to acute cold exposure. These results can lead to future studies of the influence of melatonin on BAT, in animals and humans, without the need for invasive evaluation of BAT.

Pinealectomy led to a reduced acute thermogenic capacity compared with the other groups, as well as a reduced UCP1 mRNA expression.
Conclusion Melatonin deficiency apparently impairs BAT response to acute cold exposure. These results can lead to future studies of the influence of melatonin on BAT, in animals and humans, without the need for invasive evaluation of BAT.

Background:
Melatonin is a pineal hormone,produced at night, with a critical role in the synchronization of circadian rhythms, with known metabolic effects in many animal species (1,2). One of its metabolic effects in rodents is a reduction in body weight with minimal decrease in food intake, suggesting an action on energy expenditure, possibly related to activation of brown adipose tissue (BAT) (1)(2)(3)(4)(5).
Indeed, many experimental models have shown the role of melatonin on BAT recruitment (3,6) BAT is a thermogenic tissue whose primary function is thermoregulation via non-shivering thermogenesis (7). Thermogenesis occurs due to a unique and specific enzyme, called UCP-1 (uncoupling protein-1), which uncouples ATP energy production in the mitochondria, generating heat instead (8). Brown adipose tissue is activated by sympathetic noradrenergic stimuli, and cold is theimportant physiological stimulus known (7,8). 4 Recently, BAT research has increased after positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro-D-glucose ( 18 F-FDG PET) has shown that humans still possess active BAT, especially after cold exposure (9,10). In this context, a lack of BAT activation could have a possible role in weight gain in humans, and targeting BAT activation could be a weapon for the treatment of obesity and/or type 2 diabetes (11).
Many compounds have been studied in order to determine the ability of BAT recruitment and activation, including melatonin (12,13). However, in the literature, several melatonin studies conducted in animals measured by BAT mass in order to analyze BAT recruitment and activation, and some of them focused in BAT thermogenesis measured by calorimetry or cytochrome activity (3).
More recently, some studies have evaluated a possible increase in BAT and in thermoregulatory responses, using infrared thermography (5,14,15).
It is worth noting that an increase in BAT mass (recruitment), not necessarily leads to an increased thermogenic response (activation), if there is no physiological need for heat production (7,8,16). In this regard, an increase in BAT mass or in UCP-1 expression not necessarily would mean an increase in BAT uptake in 18 F-FDG PET (16,17). Concerning this, different protocols of cold exposure, or other adrenergic stimuli, could also lead to different patterns of BAT uptake by 18 F-FDG PET that does not necessarily reflect the total BAT mass of an individual (16).
As 18 F-FDG PET is the gold-standard method for the detection of BAT in humans (18), it would be relevant in experimental studies to use this methodology to understand the role of melatonin deficiency and its supplementation on BAT thermogenic responses.
In the present study, we ought to determine if melatonin deficiency in experimental models of Animals were assigned to three groups: health animals with pineal gland (C, n=6 rats); pinealectomized animals (PINX, n=7) ; and PINX rats treated with melatonin (PM,n=6), since the day of surgery . Pinealectomy was performed as previously described (19), and 1.0 mg/kg of body weight of melatonin (Sigma-Aldrich, St. Louis, MO, USA) was added to the drinking water exclusively during the dark phase and the concentration in the drinking water solution was corrected on a daily basis using the previous night ingested volume and the bodyweight of each animal.

Positron emission tomography-computed tomography (PET-CT):
Briefly a small animal imaging Positron emission tromography ccopled to a computerized tomography machine was used to follow 2-deoxy-2-[fluorine-18]fluoro-D-glucose physiological uptake in Rats (Figure X, suplemmentary appendix). Basal imaging (pre-thermogenic stimuli) and 2-days image (post-thermogenic stimuli) were performed using 18 F-FDG PET/CT.In order to determine the 18 F-FDG uptake images were performed twice, with an interval of two days, in both room temperature (23 ± 2 o C) and after an acute cold challenge. During the acute cold challenge, rats were exposed to the environmental temperature of 4 ± 2 o C for four hours, and immediately afterward, they were scanned using 18 F-FDG PET/CT. Our freezing protocol was performed according to the literature(20).
The images were performed around ZT 6 (six hours after lights on) to preserve the BAT peak activity in rats as described before (21) The animals received i.v. 2.474 mCi (91.54 MBq) of 18F-FDG and images were obtained by small imaging PET/CT (Triumph II trimodality System, Gamma Medica/Trifoil imaging).), with a field of view (FOV) of 80 mm, matrix 240x240. Images were then co-registered with CT for anatomic correlation and analyzed by at least 2 nuclear medicine specialists, using a manually drawed region of interest 6 (ROI) in the interscapular area. The ROI was used to evaluate the interscapular maximal Standard Value Uptake (Max SUV). We analyzed data from both experiments separately as well as the ratio of SUV increase (maximal SUV after a cold challenge/maximal SUV in room temperature), that was interpreted as an "acute thermogenic activity of BAT." The schematic representation of the intervention is illustrated in figure 1.

UCP-1 mRNA expression:
After the experiments, animals were sacrificed and all interscapular tissue was collected for UCP-1 mRNA analysis. BAT was maintained in dry ice and homogenized in 1 ml of Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Samples were then treated with DNase using the kit Turbo DNA-free™ (Ambion, Austin, TX, USA). After RNA separation, the total RNA was quantified by a Spectrophotometer (NanoDrop 2000, Thermo Scientific, USA). cDNA was obtained by reverse transcriptase reaction (SuperScript™ III Antisense Transcriptase, Invitrogen, Carlsbad, CA, USA) followed by quantitative analysis of genic expression was obtained by Fast Real-Time PCR (Applied Biosystems, Foster City, CA, USA). The quantitation was achieve using the UCP1 gene Ct value to a a beta-actin housekeeping using the formula: 2 ΔΔCT .The fold-increase result was obtained dividing the UCP1 expression by house-keeping genes, in order to normalize for differences in total samples.

Statistical analysis:
18 F-FDG PET data were analyzed by unpaired t-test, comparing the group in pairs using the software Prism (GraphPad Software, La Jolla California USA). In the UCP-1 expression data, we performed ANOVA using the same software.

Results:
The cold challenge experiment was useful and increased BAT Max SUV in all three animal models with statistical significance (Fig. 1A, Supllementary Appendix) In both isolated models (room temperature and cold challenge), there was no statistical difference between the three groups regarding Max SUV (Table 1a and 1b). When the ratio of increase after cold challenge compared with room temperature was analyzed (what we called "acute thermogenic capacity"), we could find that the pinealectomized group had a statistically significant reduction in this parameter (Fig. 1B left, Table 2), compared with both other groups, confirming that melatonin deficiency could impair BAT thermogenic responses (Fig. 1B right).
After sacrifice, UCP1 mRNA was analyzed, and there was a statistical difference between P group and PM group (Table 3), confirming that melatonin deficiency reduces UCP-1 gene expression and also that melatonin is able to reestablish its expression.

Discussion:
It was our aim to test if melatonin deficiency (due to surgical pinealectomy) decreased BAT activation and if melatonin replacement could revert this pattern to normal. This is the first investigation, to our knowledge, to study BAT activation by 18 F-FDG PET scan before and after melatonin treatment in animals. Although data of increased BAT mass and thermogenesis after melatonin replacement in many animal models and species have already been demonstrated (3,6,14,15,18), we found relevant to investigate if 18 F-FDG PET was able to detect those differences, in order to facilitate future research in animals as well as humans, in which indirect measures of BAT are more feasible to be detected than direct samples tissues. Indeed, the positive results of this actual study permitted 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 (19).
In both isolated models, there were no differences between the groups, although some trends could be observed, including an unexpected higher maximal SUV in pinealectomized animals in room temperature. However, the melatonin deficient group had a clear, reduced acute thermogenic response, suggesting that melatonin proficiency is critical for thermoregulation in settings of acute challenging as temperature reduction. However, maybe in 23 o C room temperature chronic exposure, which is not thermoneutral for rats, but clearly not as challenging as acute 4 o C exposure, other mechanisms of BAT activation independent of melatonin could be enough to maintain the normal thermoregulatory responses of the animal (3). In our recent human study that demonstrated an 8 increase in BAT volume and activity after melatonin replacement in a group of pinealectomized individuals (19), some individuals have reasonable high baseline BAT activity.
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 is slightly different experimental models (5,13,14,18). Therefore, although our UCP-1 results are not original, they reinforce and validate those previous results.
The observed pattern of UCP-1 expression in our model is in line with 18 F-FDG PETresults. The groups were statistically different; P group shas the lowest UCP-1 expression, which in some ways could explain the lower thermogenic capacity.
However, as already pointed out, an increase in UCP-1 RNA not necessarily means 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 even with lower UCP-1 expression, the pinealectomized group showed a normal BAT response in 18 F-FDG PETin room temperature.
Our main issue in this particular study was to evaluate BAT responses in 18 F-FDG PET. As many other different studies have demonstrated the physiological role of melatonin in body weight regulation, food intake, energy expenditure, metabolic risk factors, as well as many other parameters, we did not include those data as they would be redundant. Our group have previously shown that pinealectomy leads to a metabolic syndrome phenotype in rats and melatonin replacement reverts it (1). Melatonin have 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.
BAT physiology is very complex, and potential compounds aiming BAT increase can act in 9 recruitment, activation, or both (3,6,11). Experimental models can help us to 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 (11,20). Our finding that melatonin seems critical for the acute BAT activation induced by cold measured by 18 F-FDG PET is novel and can lead to future imaging studies in both animals and humans that will help to understand the physiological role of melatonin in relation to BAT and if melatonin could be a potential weapon for increasing melatonin recruitment and activation in humans, with potential therapeutic use in metabolic diseases. 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 (19,(21)(22).

Conclusions:
This is the first study, to our knowledge, to evaluate BAT responses to melatonin by 18F-FDG PET, which is important essential for future melatonin and circadian studies, in animals and humans, for evaluating BAT responses by non-invasive techniques.
We demonstrated that pinealectomy impairs BAT thermogenic capacity measured by 18

F-FDG PET in
Wistar rats after an acute cold challenge and that melatonin replacement reverts this impairment.
These results are in line with many experimental models that suggest that melatonin have a critical role in BAT mass and activity, as well in UCP1 expression.

Declarations
Ethics approval and consent to participate JCN is responsible for the Thematic Project in which this trial belongs and received funding.
Dr. Jose Cipolla Neto is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.