京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13168号 / 論医博第2155号 / 新制||医||1029(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 松田 道行, 教授 川口 義弥, 教授 近藤 玄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Brown (BAT) and white (WAT) adipose tissues are significant contributors to whole-body energy homeostasis. A disturbance in their metabolic function could result in the development of obesity and subsequent metabolic complications. The energy-sensing enzyme of the cell, AMP-activated protein kinase (AMPK), has been vastly studied in skeletal muscle and liver, but its role in BAT and WAT metabolism is elusive. We generated an inducible, adipocyte-specific knockout mouse model for the two AMPK β subunits (iβ1β2AKO) and found that iβ1β2AKO mice were intolerant to cold, and resistant to β3-adrenergic activation of BAT and browning of WAT. These defects in BAT activity were not due to the AMPK-ACC axis, but instead were due to compromised integrity of mitochondria. Mitochondrial morphology, function, and autophagy were all distorted in iβ1β2AKO mice, measured via transmission electron microscopy (TEM), respiration, and immunoblotting, respectively. These findings provide strong evidence that adipocyte AMPK regulates a fine-tuned program that responds to environmental and pharmacological inputs by maintaining mitochondrial integrity through autophagy and subsequent mitochondrial biogenesis in chronic settings. / Thesis / Master of Science (MSc) / Traditionally, there are two types of adipose tissue that appear and function differently. White adipose tissue (WAT) has evolved to store away energy in an efficient manner for later use. In contrast, brown adipose tissue (BAT) is a unique organ in mammals that has evolved over time to maintain body temperature. In essence, BAT has the ability to burn away calories as heat and is a promising therapeutic target to combat obesity and metabolic diseases such as type 2 diabetes. In our study, we have identified a potential factor that not only promotes BAT activity, but also promotes WAT to function more like BAT. By targeting this factor through drugs, there is potential to increase resting metabolic rate and fight the global epidemic of obesity.

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Background: Brown adipose tissue (BAT) preferentially oxidizes stored triglycerides (TAGs) to generate heat during acute exposure to cold. However, the time course of its activation is not well described as we are currently limited to BAT measurements before and after an acute stimulus. Magnetic resonance imaging (MRI) is a preferred modality to uncover such evidence, as it estimates TAG content via fat fraction (FF), and permits repeat scans in the same subject. As such, serial FF measurements in a defined BAT region of interest during a uniform whole-body temperature challenge is warranted. Objectives: The first objective of this study was to assess the pattern of change in supraclavicular (SCV) BAT and posterior neck subcutaneous adipose tissue (SAT; a region with an unestablished role in non-shivering thermogenesis) FF during a mild cold exposure in adult males. The second objective was to evaluate if indices of body composition were related to the pattern of cold-induced change in SCV BAT FF. The final objective was to assess the influence of warming immediately following cooling on these changes. Methods: Twelve males between the ages of 19 and 28 were recruited to this cross-sectional study. Users of tobacco, nicotine, and/or alcohol, those with contraindications for magnetic resonance imaging (MRI), and diseases, surgeries, and/or medications associated with thermogenesis were excluded. There were two study visits in total. During the initial visit, anthropometric measurements were carried out in triplicate (i.e. height and weight to determine body mass index (BMI), and body composition measurements (i.e. % body total fat and lean mass (kg)) were obtained using Dual Emission X-Ray Absorptiometry. Within 30 days of this initial visit, subjects attended a time course MRI session. At this visit, participants underwent standardized cold (3-hours at 18°C) and subsequent warm (30 minutes at 32°C) exposures using a water-perfused suit while lying in a 3 Tesla MRI scanner, and the temperature of the water entering and leaving the suit was recorded throughout. FF in the SCV region and posterior neck SAT was measured at defined intervals during both temperature challenges. Separate time course plots of the mean reduction in FF from baseline were constructed for the cooling and warming phases. For the first objective, the rate and magnitude of FF changes in SCV BAT and posterior neck SAT over defined time intervals were determined through calculations of slope and area under the curve (AUC), respectively. Identification of the earliest point of change from baseline, and the point at which changes were no longer different from those measured after 3 hours of cooling, were accomplished through paired comparisons using a random-slope linear mixed model with measures at 0 minutes and 180 minutes used as the reference values, respectively. A random-intercept multilevel regression model was used to define the cold-induced change in FF over time. For the second objective, a Spearman rank-order correlation assessed the association between indices of body composition (i.e. BMI and % total body fat) and indices of BAT activity (i.e. AUC and FF reduction) at time points of interest as identified by objective 1. Results: The mean±SD of BMI, LMI, and % total body fat were 24.7±2.8kg/m2, 17.6±1.6kg/m2 and 25.0±7.4%, respectively. Seven of the twelve subjects completed three hours of cold exposure (58.3%), and a further five endured at least one hour. A significant cold-induced reduction in SCV BAT FF was detected at 10 minutes following the onset of cold exposure (mean difference = -1.6%; p=0.005), and changes in FF beyond 30 minutes of cooling were similar to those measured after three hours (p<0.05). Meanwhile, the posterior neck SAT did not experience significant cold-induced changes in FF. A novel attempt at identifying a quadratic model to predict one’s BAT-specific response to a cold challenge was carried out, and the intercept, time, time2, and intraclass correlation coefficient (i.e. parameters which described the relationship between FF and time) were highly significant (p<0.001). Although every participant had a measurable decline in FF, those with a higher BMI and % body fat had a smaller magnitude of change throughout the time course. In particular, a strong negative correlation between BMI and AUC FF decline existed as soon as 10 minutes following the onset of cold (rho=-0.786), indicating that those with a lower BMI had a larger magnitude of change in SCV BAT FF at this point. Finally, warming did not visually influence the trajectory of SCV BAT FF. Limitations: Only seven of the twelve participants completed the full 180 minutes of cold exposure, which further limited the already low statistical power of this study. Moreover, complementary measures of BAT activity, such as energy expenditure, and objective measurements of shivering, such as electromyography, could not be evaluated. Conclusions: These findings suggest that significant cold-induced changes in BAT FF occur much sooner than three hours. Thus, a shorter duration of cold exposure may be considered in future studies using MRI to detect BAT activity, as this could increase the feasibility of gathering larger and younger sample populations. / Thesis / Master of Science (MSc)

Nina Varah MSc Thesis / BACKGROUND: Brown adipose tissue (BAT) has emerged as an attractive target to address the dramatic rise in obesity and non-alcoholic fatty liver disease (NAFLD) in adults and children due to its ability to clear lipids through thermogenesis when activated with cold stimulation. Cross-sectional studies have identified an inverse relationship between BAT and NAFLD in adults, although no linking mechanism or relevance in children is known. Metabolomics provides a non-invasive platform to investigate BAT physiology and its relationship with hepatic fat in an effort to identify potential targets for further investigation. PROJECT OBJECTIVES: 1) To explore the associations between the plasma metabolome and BAT in adults and children. 2) To explore the associations between the plasma metabolome and hepatic fat in adults and children. 3) To identify metabolites associated with both BAT and hepatic fat as potential linking mechanisms for further study. METHODOLOGY: We recruited 63 male and female adults aged 18 to 57 years and 25 healthy male children aged 8 to 10 years into this cross-sectional study. Study participants underwent blood work, body composition measurement (dual energy X-ray absorptiometry; DXA) and magnetic resonance imaging (MRI) - proton density fat fraction (PDFF) measurements of whole liver hepatic fat, pre- and post-cold supraclavicular fat. BAT activity was calculated as the percent change between post and pre-cold BAT PDFF with the cold stimulus consisting of a water-perfused suit maintained at 18°C for 3-hours (adult) or 1-hour (pediatric). Targeted liquid-chromatography/mass spectrometry metabolomics of 102 metabolites was conducted on fasted plasma and multivariate linear regression with multiple testing correction was used to examine metabolite predictors of BAT measures and hepatic fat. RESULTS: In the adult cohort (n=63, median age 25.9 years, median body mass index (BMI) 25.4 kg/m2), five metabolites were associated with baseline BAT lipid content, where an elevated lipid content may indicate a whiter adipose tissue-like phenotype. Aconitate and creatine commonly predict increased baseline BAT lipid content (β=0.420, P=0.001 and β=0.408, P=0.001, respectively), and reduced BAT activity (β=-0.462, P=0.002 and (β=-0.402, P=0.002, respectively). Alanine and two acyl-carnitines also predicted reduced BAT activity. Glutamic acid was similarly related to higher baseline BAT (β=0.480, P<0.001) and hepatic lipid content independent of age and sex (β=0.392, P=0.002). Three other metabolites were directly related to hepatic fat, and serine inversely. In children (n=25, median age 9.89 years, mean BMI Z-score 1.25), cysteine and cystine were trending towards a significant relationship with higher baseline BAT lipid content, and were both related to elevated hepatic fat independent of adiposity (cysteine: quadratic β=-0.714, p<0.001 and cystine: quadratic β=0.592, p<0.001). Two hydroxy-proline isomers and L-carnitine were associated with reduced BAT activity. CONCLUSION: In adults, several metabolites were associated with reduced BAT activity and with a higher baseline BAT lipid content in the non-stimulated state – aconitate and creatine were related to both. Acylcarnitines or their metabolites related to BAT in both children and adults, which may suggest areas for subsequent investigation of BAT metabolism. Glutamic acid in adults and cysteine and cystine in children were weakly related to elevated baseline BAT and hepatic fat content. Further, amino acids such as glutamic acid and cysteine may be markers of increased ectopic fat accumulation – and are also associated with a whiter ambient BAT phenotype. Cumulatively, these findings highlight targets for further investigation into BAT physiology and the link to the liver. / Thesis / Master of Science (MSc)

The large capacity of brown adipose tissue (BAT) to expend energy as heat makes it an interesting potential player in weight regulation and other metabolic conditions. This is of particular interest as it has been recognized that adult humans possess BAT. The protein responsible for the heat production is uncoupling protein 1 (UCP1), which, as the name implies, uncouples the respiratory chain from ATP production; instead heat is produced. Cold is the strongest recruiter and activator of BAT. However, also obesogenic food has a low but nonetheless significant effect on the recruitment and activation of UCP1, although the significance of this has been discussed. In the present thesis, I have studied the effect of diet on BAT and the possibilities for it to be obesity-protective. This can be done by comparing responses in wild-type mice and in UCP1-ablated mice. Since the effect of diet on BAT is low, it is of importance to control the temperature and maintain thermoneutrality. Other confounding factors to keep in mind are differences in actual energy and composition of food and also cohort differences. When controlling all the parameters mentioned and giving the mice the same obesogenic diet, the mice possessing UCP1 compared to UCP1-ablated mice had higher energy expenditure, and lower weight gain, despite eating more. This confirms the presence of a UCP1-dependent diet-induced thermogenesis. Thus, the conclusion must be that possessing UCP1 does result in obesity protection at thermoneutrality. However, the relevance for human energy balance is still not established. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 2: Manuscript. Paper 3: Manuscript.</p>

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Charles university in Prague, Pharmaceutical faculty in Hradci Králové, Department of biological and medical sciences Rheinische Friedrich-Wilhelms-University Bonn, Institute of Pharmacology and Toxicology Candidate: Michaela Čajková Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: Dr. Linda Sarah Hoffmann Title of diploma thesis: Role of Gq-coupled receptors in brown adipocytes In my diploma thesis, we focused on four Gq-coupled receptors (F2R, LPHN1, α1DAR, TSHR) in brown adipocytes (BAs), which were identified in the screen as the highest expressed in immature and mature BAs. Our goal was to validate suggestion, that Thyroid stimulating hormone receptor (TSHR) plays a key role in differentiation of BAs and that F2R, LPHN1, α1D-AR might be important for BAs. In our study, we investigated gene expression of these four receptors in BAs, using analytical methodsquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Results from analysis revealed, that expression of TSHR was increased in mature BAs, it means, that TSHR induce differentiation of BAs. The BAs transduced with short hairpin RNA (sh-RNA) against TSHR were less differentiated, this we proved also with Oil Red-O staining. Expression of adipocyte Protein 2 (aP2), peroxisome proliferator-activated...