Effects Of Activated Brown Adipose Tissue On The Metabolism In Adults And Regulation Mechanisms Of The Activity Of Brown Adipose Tissue

Metabolic syndrome (MS) is a common clinical syndrome with multiple components, such as overweight or obesity, hypertension, hyperglycemia, and dyslipidemia. Each component is affected by others in the progression of diseases, and the core link of MS is obesity and insulin resistance. With the increasing prevalence of obesity, these diseases have become the major public health problem in the world.The main cause of obesity is the imbalance of energy intake and expenditure, and the chief offender of obesity is considered as the adipose tissue. In fact, there are two types of adipose tissue:white adipose tissue (WAT) and brown adipose tissue (BAT). Early in the last century, animal studies indicated that BAT could regulate the body temperature, and then in1979, it was found that the ability of thermogenesis in BAT is owed to a mass of uncoupling protein1(UCP1) in the mitochondrial inner membrane. However, at that time, researchers didn’t think that adults have active BAT or it has important physiological function either.Until recently, with the rapid development of molecular function imaging, it was found that there is the high uptake of fluorine-18-fluorodeoxyglucose (18F-FDG) in the cervical, supraclavicular, superior mediastinal and juxtaspinal regions of adults, when people used positron emission tomography-computed tomography imaging(PET-CT) to scan the whole body. It was also confirmed by the pathologic biopsy that the tissue is BAT rather than a tumor, and because of its high biological activities the BAT was called as actived brown adipose tissue(aBAT).In2009, the New England Journal of Medicine published several independent researches that proved the existence of aBAT in adults, and those researches also indicated that the prevalence and activity of aBAT was related to age, sex, body mass index(BMI). The results suggested that aBAT is not only present in adults, but also may play an important role in the energy homeostasis. Nonetheless, previous studies were retrospective, and subjects were mostly based on patients with cancer, and the relationship between BAT and other factors were limited to the demographic characteristics. So it is important to design a cross-sectional study with large number of samples, and research the relationship between the activity of BAT and the metabolic profiles in adults, which can provide a new thinking of prevention and therapy the obesity and its related metabolic diseases.Some basic researches showed that endocrine hormones, such as thyroid and adrenal hormones, can synergistically facilitate the expression of UCP1and increase the activity of BAT. Other basic researches displayed that central neuroendocrine factors, like Neuropeptide Y (NPY) can stimulate and maintain the appetite, participate in the regulation of energy homeostasis through the hypothalamic appetite regulating network(ARN). NPY also can store in the sympathetic nerve fiber together with norepinephrine and play the neurotransmitter role in the periphery. Therefore, researchers considered that the mechanism of regulating BAT activity might be a complex network, and the central nervous system(CNS) might play a crucial role in it. Although the exact mechanism or signaling pathway has not been clearly understood, the achievements will be widely used to treat obesity in the future.PET-CT is the forerunner of the molecular functional imaging, which can not only display images of the brain, but also estimate the glucose metabolic rate of different brain regions. So we can use this technology to explore the relationship between the brain metabolism and the BAT activity, which will be beneficial to reveal the role of CNS in the regulation of BAT activity.Accordingly, we carry out a cross-sectional study in the healthy adults. The metabolic profiles of aBAT adults will be compared with their corresponding controls. The relationship between the brain glucose metabolism and the BAT activity can be analyzed by18F-FDG PET-CT. The effect of NPY on the regulation of BAT activity should be explored by basic experiments. These results may provide clues to the therapy of obesity and its related metabolic disorders.Part I Effects of Activated Brown Adipose Tissue on the Metabolism in Chinese healthy AdultsObjective:To compare metabolic profiles of healthy adults in different groups:with aBAT(BAT positive group) and without aBAT(BAT negative group), to observe the influence of aBAT on the metabolism, and to analyze the predictors of aBAT.Methods:(1) We carried out a cross-sectional study from September1st,2009to August31st,2010at PET center of Huashan hospital affiliated Fudan University. The data of subjects who used18F-FDG PET-CT to scan the whole body and drew fasting blood sample were collected. In unstimulated condition, according to the PET-CT scanning, healthy adults were grouped by the activity of BAT. Subjects with aBAT composed BAT positive group, and in the same day, subjects without aBAT but with the same gender, the similar age and BMI composed BAT negative group. The information were comprehensively analyzed, such as demographic characteristics, physical examination, glucose and lipid metabolic profiles detected by biochemistry, adipokines, inflammatory factors, neuroendocrine factors tested by enzyme-linked immunosorbent assay(ELISA), and the PET-CT imaging data.(2) In the prospective study, some healthy adults with aBAT used PET-CT to scan the whole body and drew blood again after warm exposure in a week.(3) Multiple linear regression analysis was performed to examine the relationships between the activity of BAT and metabolism profiles. The predictors of aBAT were tested by Logistic regression with the use of univariate and multivariate models. All of the subjects signed the informed consent form.Results:(1) There were105subjects with aBAT in the594518F-FDG PET-CT scans (1.77%), and79of105was the check-up healthy adults. The BAT positive group had a total of62healthy adults with complete data, and each of them had a corresponding control.(2) In both male and female, the age and BMI of BAT positive group were lower than the negative group(all P values<0.05); the level of high-density lipoprotein cholesterol(HDL-c) and enkephalin of positive group was significantly higher than their negative controls(all P values<0.001); and the total cholesterol/HDL-c ratio (TC/HDL-c),fasting insulin, the homeostasis model of assessment-insulin resistance(HOMA-IR), leptin and NPY of positive group were significantly lower than the negative group (all P values<0.05). Only in the male, the body fat rate, fasting blood glucose(FBP), tumor necrosis factor a(TNFa) and fibroblast growth factor-21(FGF-21) of BAT positive group were significantly lower than the negative group (all P values<0.05).(3) According to the multivariate linear regression model analysis, after adjusting the possible confounding factors, such as age, sex, BMI, and even the body fat rate, the significant differences of HDL-c, TC/HDL-c, leptin, NPY and enkephalin between two groups were still existed(all P values<0.05).(4) There were15subjects with aBAT accepted the warm exposure and rechecked by PET-CT. The changes of vital sign, blood glucose, and the lipid profiles before and after warm exposure were not significantly(all P values>0.05), while the NPY was significantly higher than before (P<0.001).(5) In univariate analyses, brown adipose tissue was most frequently detected in younger(P=0.001), slender (P=0.002), and locomotor(P=0.005) subjects. In multivariate analyses, two predictors (age and exercise) remained significant.Conclusion:The age, BMI, and HOMA-IR of Chinese healthy adults with aBAT is lower than BAT negative ones, while HDL-c of adults with aBAT is higher, and the differences of adipokines, inflammatory and neuroendocrine factors between groups are also significant. It is indicated that aBAT of adults may play an important role in the metabolism, and suggested that the neuroendocrine factors, such as NPY, are closely related to the activity of BAT. In addition, the age, temperature and exercise have an effect on the activity of BAT.Part II The Relationship between Neuropeptide Y and the Activity of Brown Adipose TissueObjective:To observe the peripheral effect of NPY on the BAT, and to discuss the possible regulation mechanism.Methods:(1) The levels of NPY mRNA and the expression of NPY type1,2,5receptors in different adipocytes (mature white adipocyte and brown adipocyte), and different organizations (white adipose tissue, BAT and hypothalamus of C57male mouse) were measured by real-time quantitative polymerase chain reaction(Real-time PCR).(2) We also used the immunohistochemical and western blotting to identify the expression of NPY type5receptor in the BAT. Before and after improving the activity of BAT by cold exposure, we also applied the reverse transcription PCR(RT-PCR) to detect the expression of NPY type1,2,5receptors in BAT.(3) After induced the differentiation of C3H10T1/2pluripotent stem cell into mature brown adipocyte by bone morphogenetic protein7(BMP7), five kinds of reagents were put into the mature brown adipocytes, such as saline control, NPY, NPY5R agonists, NPY5R antagonists, and NPY together with NPY5R antagonists. Adipogenic differentiation was evaluated by Oil Red-O-staining, and the level of422-aP2and the expression of UCP1were detected by western blotting. Moreover, We used the real-time PCR to observe the levels of genes related to adipocyte differentiation, BAT thermogenesis and mitochondrial synthesis in different treatment groups, such as PRD1-BF1-RIZ1 homologous domain containing16(PRDM16), Peroxisome proliferator-activated receptor-gamma co-activator-l-alpha(PGC-la), Silent mating type information regulation2homolog3(Sirt3), Cytochrome c somatic(Cycs), Transcription factor A mitochondrial(Tfam), Nuclear respiratory factor1(Nrf1), Nrf2.Results:(1) The results showed that BAT and mature brown adipocytes could express NPY, and the expressions of NPY5R in BAT and mature brown adipocytes were significantly higher than the other NPY receptors, which was confirmed by immunohistochemical and western blotting.(2) After cold exposure, the activity of BAT was improved accompanied with the down regulation of NPY5R.(3) In vitro cell experiments, it had no significant differences in adipogenic differentiation between five groups, while the expression of UCP1in the NPY5R antagonists group and/or together with NPY were significantly higher than other groups. The expression of PRDM16between groups had no significant difference(P>0.05), but the expression of PGC-1a, Sirt3, Cycs, Nrf1, Nrf2, and Tfam which related to the thermogenesis or mitochondrial synthesis, were all significantly increased in the NPY5R antagonists group.Conclusion:Neuropeptide Y can suppress the activity of BAT and brown adipocytes through its type5receptor.Part Ⅲ The Relationship between Brain Metabolism and Brown Adipose Tissue Activity in Chinese Healthy AdultsObjective:To investigate the relationship between regional brain metabolism and the BAT activity by PET-CT, and to explore its potential mechanism.Methods:(1) In the control study, we recruited80healthy adults with aBAT(BAT positive) according to the18F-FDG PET-CT scan, and the healthy adult without aBAT but with the same gender, similar age and BMI, scanning at the same day, was chosen as the negative control(BAT negative).(2) The data of brain images were analyzed by statistical parametric mapping8(SPM8), the BAT activity were measured by the Picture Archiving&Communication System(PACS) and PET-CT Viewer, and the semi-quantitative values of brain metabolism and BAT activity were calculated by these softwares. The levels of fasting plasma NPY were tested by ELISA.(3) The multiple linear regression models were used to analyze the correlation between the brain regions of metabolic, NPY and the BAT activity in BAT positive group. Results:(1) Compared with controls, BAT positive group showed significant glucose metabolic decreases mainly in the right insula(BA13a, BA13b), the right limbic lobe uncus(BA20)(the FDR-adjusted P<0.05).(2) The two samples independent t test found that the semi-quantitative values of three brain regions in positive group were significantly lower than the negative group (BA13a P=0.011, BA13b P=0.010, BA20P<0.001).(3) In multiple linear regression models, after adjusting for the age, sex, BMI, and outside temperature, it was negative correlation between the brain metabolic values and the BAT activity (all P values<0.05). However, after further adjusting for NPY level, the correlation between the BA20metabolic values and BAT activity hadn’t statistical significance (P=0.311),and the correlation between the BA13a metabolic values and BAT activity still had statistical significance (P=0.003).Conclusion:Regional brain metabolism is closely related to the activity of BAT, which is partly independent of NPY.