Development of the sympathetic nervous system (SNS) tone onto peripheral organs has been shown to be susceptible to a wide range of external factors, such as temperature. Although it was initially postulated that the sympathetic signal is uniform across the body, there is growing evidence that there can be target-specific sympathetic signals. To date, evidence for a relationship between developmental influences on SNS tone and organ function is purely correlational. An obstacle to investigating the programming of SNS permanently altering physiology is that experimental manipulations of SNS activity during development would impact multiple organ functions simultaneously, which could affect the overall health of the animal and therefore confound interpretation of the results. Here we used brown adipose tissue (BAT) as a platform to define a critical period and identify molecules that contribute to the development of SNS outflow to peripheral organs. In addition, we explored the molecular target-specificity of sympathetic neurons by performing a single-cell RNA sequencing transcriptomic analysis of adult mouse stellate ganglion (SG) in conjunction with retrograde tracing from two of its targets, brown adipose tissue and forelimb. We discovered four molecularly distinct populations of SG neurons that express unique combinations of neuropeptides and receptors, but we did not find evidence of target specificity. The four distinct SG neuronal populations had marker genes that showed unique expression in each population, including genes encoding secreted peptides and receptors of circulating factors. Also, we found that the expression of some of the marker genes differs across the sympathetic chain, which could provide a means for coordinated regulation of SNS responses to specific types of homeostatic challenges.

Historical experiments regarding the influence of low ambient temperatures were often accompanied with the development of detrimental effects. Our laboratory recently published a protocol of mild cold acclimation (5 weeks in 8 ± 1 řC) which induces cardioprotective phenotype in rats (Rattus norvegicus). An important mechanism of cold acclimation is the activation and increase of brown adipose tissue. Besides the nonshivering thermogenesis brown adipose tissue can produce a multitude of autocrine, paracrine, and endocrine factors which might positively influence whole-body metabolism and function of other important organs. The effect of cold acclimation on brown adipose tissue is not however explored very well. The aim of this work was to asses selected parameters of respiration of isolated brown adipose tissue mitochondria of control rats living in 24 ± 1 řC and compare it with that of rats exposed to 8 ± 1 řC for 1 day, 3 days, 10 days, and 5 weeks. Results of this works were 1) the increase in respiration occurs after the 1-day cold exposure and 2) the highest respiration per 1 mg of mitochondrial protein appears to be after the 10 days of cold exposure. Key-words: Brown adipose tissue, mitochondria, respiration, cold acclimation

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20968号 / 医博第4314号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 上杉 志成, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Obesity is a growing problem that impacts both adults and children. Obesity is linked to the development of unfavorable health outcomes like excess fat accumulation in the liver, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT), a thermogenic body fat that can be turned on by cold, produces heat by consuming circulating lipids and glucose in a futile cycle. Less active cold-stimulated BAT is linked to obesity and type 2 diabetes in adults but whether this relationship exists in children is unknown. In rodents, increases in BAT activity are associated with reductions in NAFLD, effects that may be mediated through changes in the gut microbiome and reductions in peripheral serotonin. Whether the gut microbiome and serotonin play a role in regulating BAT activity in adults and children is not known. In this thesis, we have utilized magnetic resonance imaging (MRI) proton density fat fraction (PDFF) to assess BAT in the supraclavicular (SCV) region after three hours and one hour of whole-body cold exposure in adults and children, respectively. In 60 adults (aged 18-57 years), we examined whether there is a relationship between cold-stimulated BAT activity and liver fat (assessed via MRI) and whether gut microbiota plays a role in connecting these two tissues. In children, we investigated, BAT activity after whole-body cold exposure in 26 boys (aged 8-10 years). We also explored if BAT activity was different between boys with and without overweight/obesity. Finally, in young boys, we measured the levels of serotonin in platelet-poor plasma and its metabolic end product 5-hydroxyindole acetic acid (5-HIAA) in the urine. We explored if these measures of circulating serotonin were related to cold-stimulated BAT activity and if they were different between boys with and without overweight/obesity. The findings from this research indicate that higher cold-stimulated BAT activity is associated with lower liver fat in adults, but that this relationship is unlikely mediated through changes in the gut microbiota. Additionally, boys with overweight/obesity have lower cold-stimulated BAT activity and lower 5-HIAA in their urine compared to those with normal weight. Moreover, circulatory serotonin is negatively related to total adiposity. However, circulating serotonin is not related to cold-stimulated BAT activity in this cohort. These findings are important as they indicate for the first time that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity. / Thesis / Doctor of Philosophy (PhD) / Obesity is a risk factor for the accumulation of extra liver fat, a problem known as non-alcoholic fatty liver disease (NAFLD). Brown adipose tissue (BAT) is a kind of body fat that rather than storing calories like white fat burns calories when switched on by cold. Studies in adults have shown that people with obesity and type 2 diabetes have less active BAT suggesting switching it on may be helpful to promote weight loss and lower glucose. However, whether this relationship exists in children is not known. In rodents, increased BAT activity has also been linked to reductions in NAFLD, effects that might involve a hormone called serotonin, or changes in the gut microbiome but whether this is important in children and adults is also not understood. In this thesis, we utilized magnetic resonance imaging (MRI) to examine BAT activity after whole-body cold exposure in adults (3 hours) and children (1 hour). In 60 adults (aged 18-57 years), we report that higher cold-stimulated BAT activity is linked to NAFLD, but gut microbiota does not seem to play a role in this relationship. In 26 boys (aged 8-10 years), BAT is less responsive to cold in boys with overweight/obesity compared to boys with normal weight. Additionally, serotonin is lower in boys with overweight/obesity compared to boys with normal weight. These findings suggest that increasing BAT activity in adults and children could potentially be a new avenue for the treatment of NAFLD and obesity.

No description available.

BACKGROUND: Brown adipose tissue (BAT) is a thermoregulatory tissue that may have a positive influence on metabolic health by improving glucose homeostasis, reducing adiposity and increasing energy expenditure. It is enriched with uncoupling protein-1 (UCP1) and therefore produces heat by uncoupling oxidative phosphorylation from ATP production. It has long been known that infants are born with BAT, however, only recently has BAT been reported in children and adults. In humans, BAT is predominantly located in the supraclavicular (SCV) region, however there are smaller depots in the peri-renal and mediastinal areas. BAT has primarily been studied in humans using Positron Emission Tomography/Computed Tomography (PET/CT). Such studies have demonstrated that children appear to have a higher prevalence of BAT than adults, however this methodology is not suitable for widespread research in healthy children. Therefore non-invasive methods that accurately measure BAT are required. The factors influencing BAT activity are of interest as this tissue may act as a desirable therapeutic target for metabolic diseases such as obesity and diabetes. PURPOSE: The purpose of this thesis project was two fold; Part 1 involved the examination of SCV skin temperature as a measure of BAT activity in children and the objective of Part 2 was to determine if SCV skin temperature had a relationship to body composition and physical activity in children. METHODS: We recruited 33 pre-pubertal boys (ages 8-10) to this cross-sectional study. The study included 3 visits, in which we measured lean mass and fat mass with dual-energy X-ray absorptiometry, resting energy expenditure (REE) before and after a 30 minute 12ºC cold exposure with indirect calorimetry, objective physical activity with an accelerometer and SCV temperature (measured every 2.5 minutes during a 30 minute, 12ºC cold exposure) with an Infrared Thermal Camera. Lean mass and Fat mass were quantified as lean mass index (LMI) and fat mass index (FMI). Physical activity was quantified as total accelerometer counts per minute (CPM) and minutes of moderate to vigorous physical activity (MVPA) per day. For Part 1, we assessed the precision of IR-thermal imaging of SCV skin temperature by examining the reproducibility of eight skin temperature outcomes over two trials. Furthermore, we assessed the accuracy of these eight skin temperature outcomes by investigating their association with energy expenditure. For Part 2 we evaluated the relationship of FMI, LMI and physical activity (MVPA) with SCV skin temperature in a multivariate analysis. RESULTS: Following these analyses, post-cooling skin temperature had the highest reproducibility of the eight skin temperature outcomes (intraclass correlation coefficient of 0.95, p<0.001) and was also significantly correlated with energy expenditure (Pearson correlation=0.392 p=0.032). Therefore, we used this outcome measure when examining the relationship between SCV skin temperature, body composition and physical activity. Fat mass index (FMI) was inversely related to post-cooling SCV skin temperature (β= -0.125, p<0.001). Minutes of moderate to vigorous physical activity and lean mass index (LMI) were not related to post-cooling SCV skin temperature. CONCLUSION: This study determined that post-cooling SCV skin temperature may be useful for detecting BAT in children and it is inversely related to adiposity. / Thesis / Master of Science (MSc)

BACKGROUND: There has been renewed interest in the study of brown adipose tissue (BAT) as a potential therapeutic target for obesity, diabetes and non-alcoholic fatty liver disease (NAFLD). There is now much evidence to suggest that BAT is not only important in thermogenesis but also plays an important role in metabolism. In adults, cold-induced BAT activation has led to a significant increase in insulin sensitivity and energy expenditure as well as decreased blood sugar levels. Thus, it is important to identify factors associated with these metabolic disorders such as the presence and activity of BAT to better understand if and how BAT can be targeted to treat these disorders. However, as a potential therapeutic target, it is important to develop accurate, precise, robust and reproducible non-invasive modalities to measure BAT. PROJECT OBJECTIVES: 1) Develop and assess protocols for the use of MRI in measuring BAT characteristics and activity 2) Examine the relationship between BAT MR outcomes and known covariates such as age, sex, body fat percentage and outdoor temperature in adult humans 3) Determine if there is any association between BAT outcomes and liver fat in adult humans, before and after adjusting for potential covariates of liver fat such as age, sex and body fat percentage METHODS: In total, 36 healthy participants (i.e. no conditions or medications that could influence BAT metabolism and/or liver disease) aged 18 to 60 years were recruited to this cross-sectional study. There were two study visits. In visit 1, anthropometrics (i.e. height, weight and waist circumference), blood pressure and body composition (via dual x-ray energy absorptiometry) were measured. Additionally, fasting bloodwork was collected and a 75-g oral glucose tolerance test (OGTT) was administered. During visit 2, participants were exposed to a standardized cold exposure set at 18°C for 3 hours using a water-perfused suit. MRI scans were acquired to evaluate changes in fat-fraction (FF%) and T2* relaxation (T2*) (BAT MR outcomes), liver fat and abdominal fat after a cold exposure. During the cold exposure protocol, mean skin temperature (MST) was monitored using 12 wireless temperature loggers placed at different sites of the body while electromyography (EMG) was used to measure shivering intensity. RESULTS: In the current study, an MRI protocol capable of detecting BAT in the supraclavicular (SCV) region was developed. This protocol included the use of FF and T2* masks to more accurately characterize BAT in the SCV region. Additionally, the MR segmentation protocol was found to be very reliable, as demonstrated by excellent ICC values (i.e. ICCagreement and ICCconsistency ≥ 0.90) for all BAT MR outcomes irrespective of cold exposure. As expected, FF% (mean difference = -2.97; p < 0.0001*) and T2* (mean difference = -0.84; p < 0.0001*) values in the SCV significantly decreased after cold exposure, consistent with BAT activation. Furthermore, the decline in both FF% and T2* after cooling was specific to the SCV region, as these changes did not occur in the posterior neck fat. In examining the relationship between BAT MR outcomes and known covariates of BAT (i.e. age, sex, body fat percentage and outdoor temperature), it is important to note that lower FF% or T2* values are reflective of a browner phenotype while a greater reduction in FF% is indicative of higher BAT activity. BAT characteristics (A: pre-cold FF%; B: pre-cold T2*) and BAT activity (C: FF% reduction) were correlated with age (A: r = 0.54; p = 0.0007*; B: r = 0.42; p = 0.0112*; C: r = -0.39; p = 0.0213*) and body fat percentage (A: r = 0.83; p < 0.0001*; B: r = 0.58; p = 0.0002*; C: r = -0.64; p < 0.0001*). That is, higher age and body fat were associated with a less brown phenotype prior to cold exposure and with less BAT activity (i.e. lower FF% decline) in response to cold exposure. However, no associations were found between BAT MR outcomes and sex or outdoor temperature. Lastly, liver fat was associated with higher values of pre-cold FF% (r = 0.60; p < 0.0001*) and pre-cold T2* (r = 0.47; p = 0.0040*) while FF% reduction was inversely correlated with liver fat (r = -0.38; p = 0.0295*). Additionally, the relationship between BAT MR outcomes and liver fat still existed after adjusting for age and sex while its effects were mediated by adiposity. CONCLUSION: In this study, a highly reliable MR segmentation protocol was developed that is capable of measuring BAT characteristics and activity irrespective of cold exposure. Additionally, the cold exposure protocol used was sufficient to elicit changes in BAT MR outcomes, as demonstrated by significant changes in FF% and T2* after cooling. Consistent with previous studies, BAT outcomes (as measured by MRI) were associated with age and body fat percentage. Lastly, findings in this thesis provide strong supporting data that BAT may regulate liver lipid content, however, the extent and mechanisms remain to be determined. / Thesis / Master of Science (MSc)

Résumé : Au Canada, un tiers des enfants souffrent d’embonpoint ou sont obèses. Les événements survenant au cours de la vie intra-utérine jouent un rôle important dans la détermination de la susceptibilité des enfants à développer des maladies liées au métabolisme énergétique comme l’obésité et le diabète. L’épigénétique pourrait expliquer en partie ce risque à long terme. Mon projet de maîtrise s’appuie sur les résultats d’une approche à l’échelle du génome qui a pour objectif d’identifier les changements épigénétiques (méthylation de l’ADN), chez les nouveau-nés, associés à une exposition fœtale à l’obésité maternelle ou au diabète gestationnel (DG). Cette stratégie a permis d’identifier le gène PRDM16 qui joue un rôle clé dans le développement du tissu adipeux brun (BAT). Un déficit dans la fonction du BAT résulte en une accumulation de lipides dans le tissu graisseux ce qui est associé au développement de maladies métaboliques. Notre hypothèse est que le profil de méthylation de l’ADN de PRDM16 et d’autres gènes centraux impliqués dans la genèse du BAT est perturbé par une exposition aux débalancements métaboliques associés à l’obésité maternelle et au DG. Cette étude inclut 133 femmes et leur nouveau-né recrutés dans la région du Saguenay-Lac-St-Jean. Les données anthropométriques et métaboliques des femmes ont été récoltées à la fin de chaque trimestre de grossesse. Une hyperglycémie orale provoquée (HGOP, 75g), entre la 24e et la 28e semaine de grossesse, a permis d’établir le diagnostic de DG selon les critères de l’OMS. À la naissance, des biopsies de placenta ont été recueillies ainsi que les données anthropométriques et métaboliques pour chaque enfant. Les niveaux de méthylation de l’ADN des gènes PRDM16, PPARGC1α, BMP7 et CTBP2 dans le placenta fœtal ont été mesurés par pyroséquençage de l’ADN traité au bisulfite de sodium. Les résultats ont montré que le DG était associé à une méthylation de l’ADN du gène BMP7 plus faible dans le placenta des nouveau-nés exposés au DG contrairement à ceux non exposés. Les niveaux de méthylation des gènes BMP7, PPARGC1α et PRDM16 étaient corrélés au statut glycémique maternel au 2e trimestre de grossesse et expliquaient une partie des niveaux de leptine dans le sang de cordon ombilical. Ces résultats suggèrent que la méthylation des gènes associés à la genèse du BAT est affectée par le DG. Ces résultats suggèrent également que le métabolisme énergétique des enfants exposés au DG est altéré ce qui pourrait mener au développement de troubles métaboliques plus tard dans la vie comme l’obésité et le diabète de type 2. // Abstract : In Canada, one third of children are overweight or obese. The events occurring during the intrauterine life play an important role in determining the susceptibility of children to develop diseases related to energy metabolism such as obesity and diabetes. Epigenetic changes can to explain this long-term risk. My project is build on the results of an epigenome-wide aiming to identify epigenetic changes (DNA methylation) in newborns associated with fetal exposure to maternal obesity and gestational diabetes (GD). This strategy has identified the PRDM16 gene which plays a key role in the development of brown adipose tissue (BAT). A deficiency in the function of BAT results in accumulation of lipids in the adipose tissue that is associated with the development of metabolic diseases. Our hypothesis is that the profile of DNA methylation at PRDM16 gene and other genes involved in BAT genesis are disrupted by exposure to metabolic dysregulation associated with maternal obesity and GD. This study included 133 women and their newborn recruited in the Saguenay-Lac-St-Jean region. Anthropometric and metabolic data of the women were collected at the end of each trimester of pregnancy. An oral glucose tolerance test (OGTT, 75g), between the 24th and 28th week of pregnancy, resulted in the diagnosis of GD according to the WHO criteria. At birth, the fetal placenta biopsies were collected and anthropometric and metabolic data for each newborn. DNA methylation levels of PRDM16, PPARGC1α, BMP7 and CTBP2 genes in fetal placenta were measured by pyrosequencing of sodium bisulfite treated DNA. The results showed that GD was associated with lower DNA methylation of BMP7 gene in the placenta of newborns exposed to GD in contrast to those not exposed. DNA methylation levels of BMP7, PPARGC1α and PRDM16 genes were correlated with maternal glycemic status in the 2nd trimester of pregnancy and partially explained leptin level variability in cord blood . These results suggest that DNA methylation of genes associated with BAT genesis is affected by GD. Therefore, energy metabolism of children exposed to GD could be altered and thus leading to the development of metabolic disorders such as obesity and type 2 diabetes later in life .

Mitochondria play a central role in lipid metabolism and pathology in obesity and type 2 diabetes mellitus. Mitochondria have been shown to associate with lipid droplets (LDs) in multiple tissues but the functional role of these peridroplet mitochondria (PDM) is unknown. This work reveals that PDM have unique protein composition and cristae structure, and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities. (2) PDM have reduced beta oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and beta oxidation. (3) PDM support LD expansion as Perilipin 5-induced recruitment of mitochondria to LDs increases ATP-dependent triacylglyceride synthesis. (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis. We suggest that increased mitochondrial recruitment to LDs may be part of a generalized adaptive response in physiological conditions that require LD expansion, such as post-prandial lipid synthesis and storage. Furthermore, PDM-mediated LD expansion may play a role in muscle and liver injury from lipotoxicity in conditions of nutrient excess, such as obesity and hyperlipidemia. A better understanding of PDM and LD biology may therefore lead to new therapies for lipotoxic tissue injury and insulin resistance. / 2020-10-24T00:00:00Z

O peso corporal dos animais tende a ser relativamente estável durante longos períodos de tempo. Situações de restrição calórica ou aumento na ingestão de calorias levam a alterações fisiológicas compensatórias que resistem aos efeitos destas perturbações. De fato, o gasto energético aumenta em animais submetidos à dieta hipercalórica, a chamada termogênese facultativa, de modo a manter os estoques energéticos constantes. É possível que defeitos na termogênese facultativa estejam envolvidos no desenvolvimento da obesidade. O BAT, o principal sítio de termogênese facultativa, é ativado pela liberação de NE pelo Sistema Nervoso Simpático, que se liga aos receptores adrenérgicos b1, b2 e b3 expressos nos adipócitos marrons. Diversos estudos demonstram que os receptores b são importantes na proteção contra a obesidade, mas ainda não é claro qual o papel de cada isoforma neste processo. Assim sendo, o objetivo do nosso trabalho foi avaliar o papel das isoformas b1 e b2 na mediação da termogênese facultativa induzida pela dieta. Para tanto, nós tratamos camundongos com nocaute para o receptor adrenérgico b1 (KOb1) e camundongos com nocaute para o receptor b2 (KOb2) com dieta hipercalórica por 22 semanas. O peso corporal foi medido diariamente e o consumo de oxigênio foi determinado usando-se um sistema de respirometria aberto ao final do experimento. A composição corporal foi determinada pela análise da carcaça. Animais foram expostos ao frio de 4ºC por 4h e sua temperatura corporal foi medida em vários tempos e a resposta térmica do iBAT foi determinada pela infusão de NE ou agonista b adrenérgico. Além disso, foram determinados os níveis de RNAm das isoformas de receptores adrenérgicos b nos animais nocaute. Os resultados obtidos em nosso estudo mostram que os animais KOb1 e KOb2 tratados com dieta hipercalórica não desenvolvem obesidade mais severa do que os animais selvagens mas não são capazes de aumentar o consumo de oxigênio induzido pela dieta, sugerindo que estes receptores não são relevantes na termogênese induzida pela dieta. Por outro lado, nossos dados indicam que a presença do receptor b1 é exigida para termogênese induzida pelo frio, uma vez que os camundongos KOb1 são sensíveis ao frio e a capacidade termogênica do BAT destes animais em reposta à NE é bastante reduzida quando comparados com animais selvagens. A ausência do receptor b2 não piora a resposta dos animais ao frio sugerindo que esta isoforma não esteja envolvida na termogênese induzida pela dieta ou pelo frio. Os nossos achados indicam que a isoforma do receptor adrenérgico b1 é fundamental na termogênese induzida pelo frio, mas não pela dieta. Além disso, é provável que a termogênese induzida pela dieta seja regulada por mecanismos distintos da termogênese induzida pelo frio. / The body weight of animals tends to be relatively stable over long periods of time. Situations of caloric restriction or increase in intake of calories lead to compensatory physiological changes that resist the effects of these disorders. In fact, the energy expenditure increases in animals treated with diet hypercaloric called facultative thermogenesis, in order to keep to energy stock constant. Defects in this facultative thermogenesis may be related to the development of obesity. Brown adipose tissue is the main site of facultative thermogenesis and is activated by signaling of b1, b2 e b3 adrenergic receptors by Norepinephrine released by Sympathetic Nervous System. Several studies showed that the isoforms b of adrenergic receptors are important in mechanisms involved in obesity and also in promoting cold tolerance. Nonetheless, it is unclear the role of each isoform in these process. Therefore, the purpose of our study was to evaluate the role of isoforms b1 and b2 in mediate the facultative thermogenesis. For that, we fed nocaute mice for the adrenergic receptor b1 (KOb1) and nocaute mice for the adrenergic receptor b2 (KOb2) with high fat diet for 22 weeks. During treatment body weight was determined daily. By the end of the experiment oxygen consumption was measured using a system of open respirometry and body composition was determined by analysis of the carcass. We also exposed KOb1 and KOb2 animals to cold (4C). The thermogenic response of iBAT was evaluated through i.v NE infusion. The results obtained in our study showed that the animals KOb1 and KOb2 treated with high fat diet did not gain more fat when compared to wild type animals, but are unable to increase the oxygen consumption, suggesting that these receptors are not relevant in development of obesity. Furthermore, our data indicate that the presence of the b1 receptor is required for cold-induced thermogenesis, since the KOb1 mice are sensitive to cold and BAT thermogenic response is significantly impaired when compared with animals wild type. The absence of b2 receptor does not worsen the response of animals to cold suggesting that this isoform is not involved in the diet- or cold- induced thermogenesis. In conclusion, our findings indicate that the b1 isoform of the adrenergic receptor is critical in the cold-induced thermogenesis, but not in diet induced thermogenesis. Moreover, it is likely that the diet-induced thermogenesis and cold-induced thermogenesis are regulated by different mechanisms.