brown adipose tissue ( bat ) is a kind of fat tissue different from white adipose tissue ( wat ) . in mammals
, bat has its function mainly in nonshivering thermogenesis , responsible for control of body temperature and regulation of energy balance .
bat is commonly found in human newborns and small mammals , and it is previously thought that there is only a vestigial amount of bat in adulthood .
recently , f - fdg pet / ct demonstrated a relatively broad distribution of functional bat in adult human , mainly along the way of sympathetic nerve control , such as paravertebral and periadrenal regions [ 3 , 4 ] .
these functional bats have drawn our attention as a potential therapeutic target for inducing weight loss through its energy expenditure pathway [ 2 , 5 ] , because excision or denervation of interscapular bat can produce abnormal increase in the amounts of wat in those animals , meaning that they are becoming fatter .
some other studies , though showing limited success , have already tried to activate bat by stimulating sns to control body weight , using , for instance , 3-adrenoceptor agonists cl-316243 or l-796568 [ 7 , 8 ] . but
cold stimulus has been proved to increase f - fdg uptake , an indicator of bat metabolism , in either animal or human studies [ 9 , 10 ] .
cold can activate sympathetic nerve system ( sns ) to excrete norepinephrine , and then the bat , which is rich in sympathetic nerve terminals , will produce more heat .
genomic studies have indicated that the heat production is related to uncoupling protein 1 ( ucp1 ) , which is highly expressed in the mitochondria of bat cells and can convert glucose and free fatty acid into heat .
for further evaluation of how the influence factors related to sns changed the bat metabolism , this study investigated several interventions to stimulate or inhibit sns and bat metabolism of mice , and under the application of micropet , bat metabolic statuses were clearly seen in vivo , showing different degree of fdg uptake under different interventions which could be semiquantitatively analyzed in real time .
micropet served as a useful tool in monitoring bat function under different interventions and showed the potential to guide further study of the relationship between bat metabolism and obesity and diabetes .
five - to - ten - wk female kunming mice ( provided by beijing medical college animal breeding center ) were enrolled in this study ( mean body weight = 25  g ) .
all animals were kept in the animal breading center of pumch with constant room temperature of 21c .
control mice ( n = 12 ) were examined under room temperature of 20 - 21c .
three intervention groups were involved in this study ( table 1 ) . in physical intervention group , mice were exposed to cold ( n = 6 , 6c-7c , t = 1  h ) , cold and then warm ( n = 6 , 6c-7c , t = 1  h , and then 35c-36c , t = 1.5  h ) , or only warm ( n = 6 and t = 1.5  h ) . for pharmacological stimulation group , under 21c mice received intraperitoneal injection of norepinephrine ( ne , 0.4  mg / kg , n = 5 ) , epinephrine ( 0.02  mg / kg ,
n = 5 ) , isoprenaline ( 0.016  mg / kg , n = 6 ) , respectively , or both cold and ne ( ne : 0.04  mg / kg , n = 7 ) . for pharmacological inhibition group ,
cold pre - exposure mice received intragastric administration of propranolol ( 13.2  mg / kg , n = 3 ) or saline ( n = 3 ) . control mice were kept in the examination room with constant temperature of 20c-21c without physical intervention or pharmacological stimulation . for cold intervention , we put the mice in a plastic box and kept them in a temperature - regulative refrigerator ( bc-185fa , aucma , china ) with a constant temperature of 6c-7c ( measured by a thermometer in the refrigerator ) for 1  h. for warm stimuli , we placed the mice in a plastic box laid above a power - regulative heating pad ( ml-1.5 - 4 , tianjin tester company , china ) with the temperature of 35c-36c ( measured by a thermometer in the box ) for 1  h and kept warming the mouse for 0.5  h after fdg injection .
norepinephrine ( tianjin jinhui amino acids co. ) , epinephrine ( beijing yongkang phar co. ) , and isoprenaline ( shanghai harvest phar co. ) were diluted with saline and injected into peritoneal cavity of mouse in the volume of 100  l ( 0.4  mg / kg ) , 100  l ( 0.02  mg / kg ) , and 30  l ( 0.016  mg / kg ) , respectively , 1  h before fdg injection .
propranolol tablets ( tianjin lisheng phar co. ) were dissolved in saline to 1  mg / ml and administered intragastrically through a small animal lavage needle in the volume of 300  l ( 13.2  mg / kg ) per mouse 1  h before fdg injection . to monitor the combination effect of cold stimuli and ne
, we performed the cold stimuli protocol as stated and gave ne injection with 0.04  mg / kg 1  h before fdg injection .
all of these pharmacological interventions were performed under room temperature of 21c , and after interventions , water and food were provided .
after physical or pharmacological interventions , 3.7  mbq f - fdg was injected into peritoneal cavity for each mouse .
anesthesia using summit as-1 - 000 - 7 animal anesthesia system ( usa ) was performed with 1.5% isoflurane ( with o2 combination of 2 liter per minute ) 40  min later .
pet data acquisition procedure was performed under siemens inveon system 10  min after anesthesia when mice were totally unconscious .
anesthesia was continued during the scanning process with the same flow rate through a facemask designed for small animal .
all micropet images were studied by two researchers ( wu and cheng ) on a high - resolution computer screen applying asipro vm software .
, the two researchers compared the interscapular bat f - fdg uptake color intensity of mouse from different intervention groups . for semiquantitative analysis ,
3d round regions of interests ( roi ) were placed carefully over the interscapular bat and brain on micropet images for each mouse , respectively .
f - fdg uptake value was acquired in the form of nanocurie per cubic centimeter ( nci / cc ) automatically after placing rois .
uptake ratio ( r ) of maximum interscapular bat uptake and mean brain uptake was calculated for each mouse and compared between different interventions .   data were reported as means  sd .
in visual analysis , different interscapular bat f - fdg uptake intensity was found under different interventions as demonstrated by the micropet images ( figure 1 ) .
cold and ne exposure caused the highest bat uptake , followed by cold exposure and the control , while propranolol lavage and warming up showed decreased bat uptake in cold pre - exposed mice .
in semiquantitative analysis , bat f - fdg uptake was significantly higher under cold exposure compared to control mice under room temperature ( r : 10.22  4.13 versus 4.08  1.32 , p = 1.96  10 ) and highest with both cold exposure and ne stimulations ( r : 15.64  5.58 versus 4.08  1.32 , p = 2.19  10 ) .
a stimulation with only ne ( r : 10.55  5.85 ) , epinephrine ( r : 4.57  0.86 ) , or isoprenaline ( r : 4.64  2.67 ) at room temperature all increased bat uptake compared with the controls ( r : 4.08  1.32 ) , but only ne showed significant difference compared with the controls ( p = .002 ) ( figure 2 ) . for bat inhibition , warming could significantly reduce bat uptake in mice with cold pre - exposure ( r : 2.13  0.43 versus 10.22  4.13 , p = .001 ) and without cold pre - exposure ( r : 2.48  0.88 versus 4.08  1.32 , p = .017 ) .
in addition , propranolol could significantly reduce bat uptake in cold pre - exposed mice ( r : 1.30  0.16 versus 3.09  0.90 , p = .027 ) ( figure 3 ) .
brown adipose tissue ( bat ) has been shedding new light on weight control of human recently , but few researches focused on monitoring bat metabolic function using molecular imaging .
it is well known that bat has more sympathetic innervations than wat , which means that bat may be activated by stimulating sns . for future investigation of new methods to control obesity through activation of bat function , the present study aimed mainly at establishing a technology platform using micropet to evaluate the bat function through interventions with factors related to sns activation or inhibition . considering that the sns might probably be modulated by short - term interventions , the temperature control or drug administration was generally set as 1  h before fdg injection in this study . moreover ,
bat can bring in false positive results in pet images of human by showing increased radioactive uptake especially in the supraclavicular area [ 14 , 15 ] ; many studies , therefore , try to find methods to inhibit bat uptake in humans , for instance , by keeping the patients warm or giving orally propranolol administration [ 1618 ] .
our study provided new data concerning these two methods in inhibiting bat uptake that only 1.5 hour of warm intervention ( 1  h before and 0.5 hour after fdg injection ) could quickly and markedly inhibit bat metabolism ( r : 2.48  0.88 versus 4.08  1.32 , p = .017 ) , and compared with propranolol intervention , warming seemed to be more effective in reducing bat activity ( p = .001 ) . based on this result , in clinics , to prevent bat uptake in pet images , providing a warm environment for patients right before pet scan would be a more effective , convenient , and safer way than propranolol intake .  
cold stimulus was proved useful to increase bat metabolism in either animal or human studies [ 9 , 10 ] because it can activated sympathetic nerve terminals surrounding the bat to excrete ne for more heat production . in our study ,
cold exposure also significantly increased bat activity ( r : 10.22  4.13 versus 4.08  1.32 , p = 1.96  10 ) , and this effect was more obvious when combined with ne injection ( p = 2.19  10 ) , well confirming that bat metabolism was correlated with catecholamine system . moreover ,
cold effect could be quickly suppressed by warming ( r : 2.13  0.43 versus 10.22  4.13 , p = .001 ) or propranolol administration afterwards ( r : 1.30  0.16 versus 3.09  0.90 , p = .027 ) , both of which were antagonistic to sympathetic - adrenal system , thus prohibiting bat activity .   in our present study the -adrenoceptor agonists ( ne , epinephrine and isoprenaline )
all increased interscapular bat activity in mouse , but only ne showed a significant difference to controls ( p = .002 , .459 , and  .293 , resp . ) .
we think the dosage insufficiency of epinephrine and isoprenaline we gave mice may answer this question . with limited researches and few examples
to follow , we gave a tentative dosage of epinephrine ( 0.02  mg / kg ) and isoprenaline ( 0.016  mg / kg ) to mice , which is about the clinical dosage for children ( about half of adult dose ) , much smaller than the advised dose for mouse ( about 9-fold clinical dosage for adults ) based on surface area per kilogram .
however , the dosage of ne ( 0.4  mg / kg ) we gave mice was 11 times the dosage for human adult , so only ne was effective in activating bat metabolism .
baba et al .   used epinephrine in 5  mg / kg , and micropet scan showed significant bat uptake compared to controls ; therefore , we think increasing dosage in future study may overcome this problem .   there are some limitations in this study .
for instance , only female mice were enrolled in this study , and the mouse number in each intervention group was not big enough ( n = 3 ~ 7 ) .
furthermore , some uncontrollable factors such as environmental noise and stimuli caused by experimental handling may potentially influence the nerve system of mouse and thus the activity of bat . in future studies
in general , this preliminary study gives new prospective in studying the stimulation and inhibition of bat of mouse by a quantitative analyzing tool of micropet . by applying micropet ,
environment temperature control can significantly stimulate or alleviate bat uptake of f - fdg within 1  h in mice . stimulating sympathetic nerve system by norepinephrine can significantly increase the metabolism of bat within 1  h , while inhibiting sns by warming or propranolol can alleviate bat function . this preliminary study with f - fdg micropet warrants further investigations of the mechanism of bat and various methods of intervention according to clinical purposes .
however , better optimization of different intervention conditions as well as some other methods for activating bat should be further explored and studied in the hope of future human application .
 brown adipose tissue ( bat ) is emerging as a potential target for treating human obesity . 
 it has been indicated that bat is rich in innervations of sympathetic nerve control . using 18f - fdg micropet imaging 
 , this study aims at evaluating how factors related to sympathetic activation / inhibition changed bat metabolism of mice . 
 bat 18f - fdg uptake were semiquantitatively evaluated in different groups of mice under temperature ( cold or warm stimulus ) or pharmacological interventions ( norepinephrine , epinephrine , isoprenaline , or propranolol ) and were compared with the corresponding controls . 
 it was found that bat activation can be stimulated by cold exposure ( p = 1.96  104 ) , norepinephrine ( p = .002 ) , or both ( p = 2.19  106 ) within an hour before 18f - fdg injection and can also be alleviated by warming up ( p = .001 ) or propranolol lavage ( p = .027 ) . 
 this preliminary study indicated that bat function could be evaluated by 18f - fdg pet imaging through short - term interventions , which paved the way for further investigation of the relationship between human obesity and bat dysfunction . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion