Study Of Thermogenic Activity And Uncoupling Protein-1Content In Brown Adipose Tissue Of Tree Shrews, Tupaia Belangeri

The adaptive thermogenesis and its regulation mechanism of mammals indifferent environment are now one of the hot topics in the study of physiologicalecology. Brown adipose tissue (BAT) is the main organ of adaptive thermogenesis,and its heat production capacity mainly depends on its molecular markers protein onmitochondrial inner membrane: uncoupling protein1(UCP1), the increase of UCP1expression is main molecular mechanism of heat production ability of the BAT. Inthis paper, we used the tree shrew (Tupaia belangeri) as the research object, whichdistributes in tropical or subtropical of south China. BAT heat production activity andUCP1content from individual, organization and molecular level were measuredunder different environment, which provide a theoretical basis for the physiologicaland ecological adaptation of small mammals in this region. Moreover, these data mayprovide some physiological and ecological basis for the treatment of obesity.This thesis mainly consists of five parts:(1) The separation and purification of UCP1from BAT in tree shrews andamplification and analysis of UCP1gene sequenceThe single component of UCP1content was successfully obtained from BAT intree shrews with hydroxylapatite column and silver staining method. Afterresearching with the method of Coomassie brilliant blue staining and silver staining,we found that the sensitivity of the protein color was significantly improved by silverstaining method, and the protein color effect is aslo better than that with anothermethod. Compared with marker protein, the results reveal that UCP1molecularweight in BAT of tree shrews was about32kDa, which were composed of about306amino acid residues, moreover, UCP1in the tree shrews were very similar to othermammals’ in the structure and function, which was consistent with previous researchresults.This experiment successfully obtained444bp BAT UCP1cDNA core fragmentof tree shrews, and we speculated that the amino acid sequence were148amino acids. After the comparison of amino acid sequence homology, the results showed that theUCP1homology between tree shrews and other animals like Mus musculus, Rattusnorvegicus, Cricetulus griseus, Oryctolagus cuniculus, Equus caballus, Felis catus,Bos taurus, Odobenus rosmarus, Pongo abelii and Ailuropoda melanoleuca wasrespectively80%,81%,82%,86%,85%,86%,89%,87%,83%and88%. UCP1coding region showed higher conservation in mammals, suggesting that UCP1playsan important physiological role in vertebrates.(2) The seasonal changes of BAT heat production activity and UCP1content intree shrewsIn the seasonal changing, body mass, body fat mass, BAT mass, liver mass,resting metabolic rate (RMR), nonshivering thermogenesis (NST) and food intake oftree shrews in winter were significantly higher than that in summer; the length andweight of the stomach, small intestine, and large intestine were maximum in winter,and minimum in summer; serum leptin level in summer were significantly higherthan that in winter; the changes of UCP1content in tree shrews were differentsignificantly in different seasons, and its seasonal variation trends were highest inwinter and lowest in summer, then higher in autumn and lower in spring. Theseresults may indicate that UCP1plays an important role in the seasonal thermogenesisregulation and energy metabolism in tree shrews.(3) Effects of cold acclimation on BAT thermogenic activity and UCP1contentin tree shrewsDuring cold acclimation, thermogenesis of tree shrews increased significantly,and compared with the control group (0d), the body mass, RMR, NST, food intake,BAT mass, liver mass, mitochondrial protein content (MP), cytochrome c oxidaseactivity (COX) and UCP1content improved significantly in the cold acclimationgroup, however, serum leptin level decreased significantly, and apparently, UCP1content was significantly positively correlated with BAT mass and NST. Thesechanges showed that the BAT and liver plays an important role in the process of coldadaptive thermogenesis in tree shrews, which maintained constant body temperatureby increasing energy intake and heat production ability. After cold acclimation, theBAT color of tree shrews obviously deepened, and BAT mass increased by53.85%. These results indicated that the tree shrews through promoting BAT tissue hyperplasia,increasing BAT mitochondria and GTP binding ability，that is to say, UCP1content,and inducing UCP1mRNA expression upregulation to enhance the vitality of BATthermogenesis, which may be an adaptive strategy for larger diurnal temperaturedifference of habitat. Combining with the role of BAT in body mass regulation andthe status of UCP1in BAT thermogenesis, we speculate that the BAT may be used astarget organ and an energy approach for the treatment of obesity.(4) Effects of photoperiod on BAT thermogenic activity and UCP1content intree shrewsThe body mass, RMR, NST, food intake, BAT mass, liver mass, MP, COX,3,3′5-triiodothyromine (T3) and UCP1content of short photoperiod group in treeshrews were higher than the long photoperiod group, but there is no difference inserum leptin level and melatonin level in two groups. The results showed that theshort photoperiod is similar to low temperature and can aslo stimulate the tree shrews’RMR, NST, liver and BAT cellular level thermogenesis to increase. But the increaseof adaptive thermogenesis induced by the short photoperiod is mainly dependent onthe activity of COX and UCP1content increase. This research aslo indicate that shortphotoperiod is likely to be a environmental signal leading thermogenesis and energymetabolism to enchance in tree shrews.(5) Effects of fasting and refeeding on BAT thermogenic activity and UCP1content in tree shrewsFasting caused a significant decrease in the tree shrews’ thermogenesis, afterfasting, RMR, NST, total COX activity (nmolO2/min/total tissue) in liver and BATand UCP1content decreased significantly, which can recover to the level of controlgroup after refeeding. It showed that liver and BAT play an important role in theprocess of hunger thermogenesis in tree shrews. Fasting induced a significantreduction in body mass, body fat mass, BAT mass and liver mass. Body mass, BATmass and liver mass can be restored to the control levels in refeeding tree shrewexcept for the body fat mass. Fasting for12h induced a rapid reduction in serumleptin content, which can not return to the control level after refeeding. There were nopost-fasting compensatory increases in food intake, and UCP1content was significantly positively correlated with BAT mass and NST. Our data indicate that treeshrews can adjust their physiological functions integratively to cope with thestarvation by the means of decreasing body mass, adaptive thermogenesis and serumleptin levels.In conclusion, the tree shrews, which originated in the tropics and extended tothe northern limit of Tupaiodes, evolved ecophysiologically adaptable characteristicswith the Yunnan plateau, thus the thermal characteristics and energy metabolismmodel not only have the characteristics of small mammals in tropical regions, but alsohave the characteristics of the northern mammals; During cold acclimation, treeshrews maintained body temperature by increasing the RMR, thermogenesiscapability of BAT and UCP1content; under short photoperiod, tree shrews increasedenergy intake, RMR, thermogenesis capability of BAT and UCP1content adapt to thechanges of environment; under the condition of fasting and feeding, they adapted toshort-term energy deficiency mainly by regulating metabolism related proteins’expression level and reducing body mass, serum leptin level and metabolic organs’mass to reduce energy consumption. The model of body mass regulation controlledby leptin was different to the typical northern mammals, which maybe responsed anew adjustment mechanism; there is a strong plasticity to different environmentamong tree shrews. The changes of BAT mass were associated with thermogenesis;combining with the role of BAT in body mass regulation and the status of UCP1inBAT thermogenesis, we speculate that the BAT may be used as target organ and anenergy approach for the treatment of obesity.