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At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript.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>

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13490号 / 論医博第2258号 / 新制||医||1059(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 渡邉 大, 教授 稲垣 暢也, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Obesity results from an excess of adipose tissue and is a major risk factor for type 2 diabetes, cardiovascular disease, and cancer. Adipose tissue exists in two main forms: white adipose tissue (WAT), which stores energy as triglycerides, and brown adipose tissue (BAT), which dissipates stored energy as heat. White adipose tissue is composed of several subcutaneous and visceral depots, each possessing distinct molecular and functional characteristics. Brown-like adipocytes can emerge in WAT depots in response to cold or beta-adrenergic stimulation. These cells have been called “beige” or “brite” (brown-in-white) cells. The reduction of obesity in mice treated with beta-adrenergic agonists is correlated with the emergence of beige cells. Beige cell development occurs most readily in subcutaneous depots, and to the least extent in visceral depots. Understanding the molecular mechanisms underlying beige cell development in different WAT depots may be important in discovering new therapies against obesity and related diseases. Our lab has previously discovered that Tyrosine Kinase 2 (Tyk2), an important mediator of cytokine signaling, promotes the development of classical brown adipose tissue. Due to the lack of functional BAT, Tyk2-knockout (Tyk2-/-) mice become grossly obese with age and develop several symptoms of the metabolic syndrome. In the present study, we have found a potential role of Tyk2 in the development of beige cells. Here, we show that mRNA expression of BAT-selective genes (UCP1, Cidea, Cox8b, and Elovl3) is significantly reduced in subcutaneous WAT of Tyk2-knockout (Tyk2-/-) mice compared to wild-type mice. Surprisingly, BAT-selective genes are induced in Tyk2-/- subcutaneous WAT by acute starvation. These findings suggest that Tyk2 is required for the development of beige cells under ambient conditions, and that the need for Tyk2 in beige cell development is bypassed during nutritional stress, a stimulus of the sympathetic response.

Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.

<p>The domestication of the pig began about 9 000 years ago and many of the existing domestic breeds have been selected for phenotypic traits like lean meat and fast growth. Domestic pigs are phenotypically very different from the ancestral wild boar that has adapted to survive in their natural environment. Because of their divergence, crosses between domestic pigs and wild boars are suitable for constructing genetic maps and Quantitative trait locus (QTL) analyses. A cross between the Large White and the European wild boar was thus initiated in the late 1980s. A major QTL for fat deposition and growth, denoted <i>FAT1</i>, was found on chromosome 4. The aim of this thesis was to further characterise the <i>FAT1</i> locus and to identify the causative gene(s) and mutation(s). We have identified new markers and constructed a high-resolution linkage and RH map of the <i>FAT1</i> QTL interval. We also performed comparative mapping to the human genome and showed that the pig chromosome 4 is homologous to human chromosomes 1 and 8. The gene order is very well conserved between the two species. In parallel we have narrowed down the <i>FAT1</i> QTL interval by repeated backcrossing to the domestic Large White breed for six generations. The QTL could be confirmed for fatness but not for growth. Furthermore, the data strongly suggested that there might be more than one gene underlying the <i>FAT1</i> QTL. Depending on which hypothesis to consider, the one- or two-loci model, the <i>FAT1</i> interval can be reduced to 3,3 or 20 centiMorgan (cM), respectively, based on the backcross experiments. In the last study we confirm the two-loci model with one locus primarily effecting abdominal fat and another locus primarily effecting subcutaneous fat. We have identified a missense mutation in the <i>RXRG</i> gene which is in strong association with the abdominal fat QTL and the mutation is a potential candidate for that locus.</p><p>Brown adipose tissue (BAT) is a specific type of fat essential for non-shivering thermogenesis in mammals. Piglets appear to lack BAT and rely on shivering as the main mechanism for thermoregulation. Uncoupling protein 1 (<i>UCP1</i>) gene is exclusively expressed in BAT and its physiological role is to generate heat by uncoupling oxidative phosphorylation. We show that the <i>UCP1</i> gene has been disrupted in the pig lineage about 20 years ago. The inactivation of <i>UCP1</i> provides a genetic explanation for the poor thermoregulation in piglets. </p>

The domestication of the pig began about 9 000 years ago and many of the existing domestic breeds have been selected for phenotypic traits like lean meat and fast growth. Domestic pigs are phenotypically very different from the ancestral wild boar that has adapted to survive in their natural environment. Because of their divergence, crosses between domestic pigs and wild boars are suitable for constructing genetic maps and Quantitative trait locus (QTL) analyses. A cross between the Large White and the European wild boar was thus initiated in the late 1980s. A major QTL for fat deposition and growth, denoted FAT1, was found on chromosome 4. The aim of this thesis was to further characterise the FAT1 locus and to identify the causative gene(s) and mutation(s). We have identified new markers and constructed a high-resolution linkage and RH map of the FAT1 QTL interval. We also performed comparative mapping to the human genome and showed that the pig chromosome 4 is homologous to human chromosomes 1 and 8. The gene order is very well conserved between the two species. In parallel we have narrowed down the FAT1 QTL interval by repeated backcrossing to the domestic Large White breed for six generations. The QTL could be confirmed for fatness but not for growth. Furthermore, the data strongly suggested that there might be more than one gene underlying the FAT1 QTL. Depending on which hypothesis to consider, the one- or two-loci model, the FAT1 interval can be reduced to 3,3 or 20 centiMorgan (cM), respectively, based on the backcross experiments. In the last study we confirm the two-loci model with one locus primarily effecting abdominal fat and another locus primarily effecting subcutaneous fat. We have identified a missense mutation in the RXRG gene which is in strong association with the abdominal fat QTL and the mutation is a potential candidate for that locus. Brown adipose tissue (BAT) is a specific type of fat essential for non-shivering thermogenesis in mammals. Piglets appear to lack BAT and rely on shivering as the main mechanism for thermoregulation. Uncoupling protein 1 (UCP1) gene is exclusively expressed in BAT and its physiological role is to generate heat by uncoupling oxidative phosphorylation. We show that the UCP1 gene has been disrupted in the pig lineage about 20 years ago. The inactivation of UCP1 provides a genetic explanation for the poor thermoregulation in piglets.

Background: Excess white adipose tissue (WAT) in humans is considered as a harmful health index. However, increased brown adipose tissue (BAT) and brown-like adipose tissue activity are associated with increased resting energy expenditure (REE) that may help to control body weight. Exercise may enhance browning formation of WAT and reduce WAT that may lead to health improvements. Aims: a) to examine the effects of physical activity on the link between peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) and fibronectin type III domaincontaining protein 5 (FNDC5) genes in muscle, circulating Irisin and uncoupling protein one (UCP1) of WAT in humans (study 1); b) to examine the relationship between UCP1 mRNA and protein expression as well as PGC-1α, peroxisome proliferatoractivated receptor alpha (PPARα) and PPARγ genes with physical activity levels in WAT of healthy men (study 2); c) to examine the effects of different types of exercise and de-training on the UCP1 mRNA and protein expression (study 3), and d) on leptin mRNA in WAT of healthy men (study 4). Method: Study 1: A systematic review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta- Analyses. Studies 2-4: The total of 46 healthy men subjected to measurements for physical activity levels, diet, anthropometry, body composition, REE, peak oxygen consumption, 1-repetition maximum and provided subcutaneous fat biopsies to determine mRNA and protein expression of six genes in one cross-sectional study and one randomized controlled trial. Results: Study 1: No link was found between PGC- 1α and FNDC5, circulating Irisin and UCP1 of WAT in response to physical activity. Study 2: The mRNA of, UCP1, PGC-1α, PPARα and PPARγ genes of WAT were not associated with physical activity levels. The UCP1 protein expression however, was negatively associated with physical activity levels. Studies 3-4: Different types of chronic exercise and de-training do not affect UCP1 mRNA and protein expression 3 and leptin mRNA in WAT. However, effect size analyses demonstrated increased UCP1 mRNA and protein expression, PPARγ and leptin in response to chronic exercise. Conclusions: There is no evidence to support the link between PGC-1α and FNDC5 in human muscle or the link between FNDC5 and circulating Irisin and UCP1 in WAT in response to exercise. There are no effects of exercise and de-training on browning formation of WAT and no link between browning formation indices and REE, body weight as well as leptin mRNA in healthy men. Further research is required to elaborate the aforementioned phenomena.

L'attention scientifique s'est récemment portée sur l'identification des mécanismes proximaux sous-tendant les compromis évolutifs;tels que les compromis existant entre croissance/reproduction et longévité. La production d'espèces réactives de l'oxygène (ROS )a été suggérée comme un candidat potentiel ,de par sa liaison étroite au métabolisme énergétique (sous-­produits du fonctionnement mitochondrial) et son caractère inévitable. Si la production de ROS excède le niveau des défenses antioxydantes, une situation de stress oxydant va en résulter et a été associé au vieillissement . Puisque la mitochondrie n'est pas uniquement la centrale énergétique de la cellule mais aussi le principal producteur de ROS, cette thèse s'est attachée à clarifier les relations entre métabolisme énergétique , fonctionnement mitochondrial et stress oxydant ; avec des études concernant l'impact d'activités coûteuses en énergie (croissance, reproduction, thermogénèse) sur l'équilibre de la balance oxydative.