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.

El objetivo principal de esta tesis se ha centrado en el estudio de las diferencias entre ratas macho y hembra en la morfología, la función y la biogénesis mitocondrial del tejido adiposo marrón (TAM) y del hígado, mediante el análisis de distintas subpoblaciones mitocondriales obtenidas a través del fraccionamiento de la población mitocondrial total. Los resultados han puesto de manifiesto que las diferencias entre géneros a nivel mitocondrial tanto en el TAM y como en el hígado podrían ser atribuidas a la existencia de una subpoblación mitocondrial altamente diferenciada en las hembras, hecho que podría ser indicativo de un proceso de biogénesis mitocondrial distinto entre ambos géneros. Los resultados sugieren la existencia de un factor común a ambos tejidos que influiría en la regulación de dicho proceso. En este sentido, las hormonas sexuales podrían ser uno de los factores candidatos responsables de las diferencias observadas en el presente trabajo. / The main goal of this thesis has been focused on the study of gender differences in the mitochondrial morphology, function and biogenesis both in brown adipose tissue (BAT) and in liver, through the analysis of the several mitochondrial subpopulations isolated by means of the fractionation of the whole mitochondrial population. Results have reflected that the gender dimorphism stated in mitochondrial population both in BAT and in liver could be attributed to the existence to more highly differentiated mitochondria in female rats, which could be the result of a different mitochondrial biogenesis process between genders. Since the existence of a common factor which influences this process in both tissues could be hypothesized, sexual hormones could be one of the main factors responsible for the differences described in the present work

Brown adipose tissue (BAT) is the main tissue involved in non-shivering heat production. A greater understanding of BAT could possibly lead to new ways of prevention and treatment of obesity and type 2 diabetes. The increasing prevalence of these conditions and the problems they cause society and individuals make the study of the subject important. An ongoing study performed at the Turku University Hospital uses images acquired using PET/MRI with 18F-FDG as the tracer. Scans are performed on sedentary and athlete subjects during normal room temperature and during cold stimulation. Sedentary subjects then undergo scanning during cold stimulation again after a six weeks long exercise training intervention. This degree project used images from this study. The objective of this degree project was to examine methods to automatically and objectively quantify parameters relevant for activation of BAT in combined PET/MRI data. A secondary goal was to create images showing glucose uptake changes in subjects from images taken at different times. Parameters were quantified in adipose tissue directly without registration (image matching), and for neck scans also after registration. Results for the first three subjects who have completed the study are presented. Larger registration errors were encountered near moving organs and in regions with less information. The creation of images showing changes in glucose uptake seem to be working well for the neck scans, and somewhat well for other sub-volumes. These images can be useful for identification of BAT. Examples of these images are shown in the report.

Recent studies suggested the persistence of brown adipocytes in adult humans, as opposed to being exclusively present in infancy. In this study, we investigated the presence of brown-like adipocytes in adipose tissue (AT) samples of children and adolescents aged 0 to 18 years and evaluated the association with age, location, and obesity. For this, we analysed AT samples from 131 children and 23 adults by histological, immunohistochemical and expression analyses. We detected brown-like and UCP1 positive adipocytes in 10.3% of 87 lean children (aged 0.3 to 10.7 years) and in one overweight infant, whereas we did not find brown adipocytes in obese children or adults. In our samples, the brown-like adipocytes were interspersed within white AT of perirenal, visceral and also subcutaneous depots. Samples with brown-like adipocytes showed an increased expression of UCP1 (>200fold), PRDM16 (2.8fold), PGC1α and CIDEA while other brown/beige selective markers, such as PAT2, P2RX5, ZIC1, LHX8, TMEM26, HOXC9 and TBX1 were not significantly different between UCP1 positive and negative samples. We identified a positive correlation between UCP1 and PRDM16 within UCP1 positive samples, but not with any other brown/beige marker. In addition, we observed significantly increased PRDM16 and PAT2 expression in subcutaneous and visceral AT samples with high UCP1 expression in adults. Our data indicate that brown-like adipocytes are present well beyond infancy in subcutaneous depots of non-obese children. The presence was not restricted to typical perirenal locations, but they were also interspersed within WAT of visceral and subcutaneous depots.

Acclimation to cold or hardening is known for many decades through its beneficial effects on human health. In contrast, sudden exposure to cold, cold shock, is a great risk of cerebral and cardiac injury, especially in the elderly. There is very little published data on the cellular and molecular mechanisms induced by cold adaptation in heart and brain. The aim of this work was to describe and compare different properties heart, liver, brain and brown adipose tissue mitochondria of rats housed at 25 ± 1 řC and at mild cold (9 ± 1 řC, 5 weeks). The high-resolution oxygraphy, spectrophotometry and Western blotting analyses were used. We found differences in the respiratory control between the heart and liver. Cold acclimation decreased activity of the Krebs cycle enzymes. Fatty acid contribution to the respiration reached the maximum in brown fat and the minimum in the hippocampus. However, further study is necessary.

1 Abstrakt Hnědá tuková tkáň je unikátní termoregulační orgán, její funkcí je přeměňovat energii ve formě protonového gradientu na energii tepelnou a zároveň snižovat efektivitu metabolismu. Její funkce se zdá být nadějí v léčbě metabolického syndromu a komplikací s ním spojených. Efekt hnědé tukové tkáně v léčbě MS však stále není objasněn a proto jsme chirurgicky odstranili tuto tkáň u potkanů, kteří trpí metabolickým syndromem, abychom objasnili důsledky extirpace na parametry metabolického syndromu. V této práci jsme sledovali aktivitu interskapulární tukové tkáně na parametry metabolického syndromu. Srovnávali jsme parametry mezi kmeny HHTg potkanů a kontrolním kmenem potkanů Wistar a pozorovali jsme nižší hmotnost potkanů kmene HHTg přes projevy ostatních parametrů metabolického syndromu, jako je dyslipidémie, inzulinová rezistence tukové tkáně, vyšší hodnoty NEMK v krvi. Zjistili jsme, že tito potkani mají významně vyšší aktivitu iBAT, jak v inkorporaci palmitátu do lipidů, tak ve spalování lipidů ve formě CO2 a zejména v nižším věku. Tato zjištění nás vedla k hypotéze, že iBAT by mohla mít zásadní vliv na parametry MS u neobézních potkanů. Extirpací iBAT u mladých potkanů, ani u potkanů v pokročilejším věku však neměla zásadní vliv na metabolismus ani na hmotnost. Dokonce došlo ke zlepšení některých...

Chronic exposure of humans or rodents to high calorie diets leads to hypertriglyceridemia and ectopic lipid deposition throughout the body, resulting in metabolic disease. Cellular lipids are stored in organelles termed lipid droplets (LDs) that are regulated by tissue-specific LD proteins. These proteins are critical for lipid homeostasis, as humans with LD protein mutations manifest metabolic dysfunction. Identification of novel components of the LD machinery could shed light on human disease mechanisms and suggest potential therapeutics for Type 2 Diabetes. Microarray analyses pinpointed the largely unstudied Hypoxia-Inducible Gene 2 (Hig2) as a gene that was highly expressed in obese human adipocytes. Imaging studies demonstrated that Hig2 localized to LDs in mouse hepatocytes and the human SGBS adipocyte cell line. Thus, this work examined the role of Hig2 as a LD protein in liver and adipose tissue. Hig2 deficiency reduced triglyceride deposition in hepatocytes; conversely, ectopic Hig2 expression promoted lipid deposition. Furthermore, liver-specific Hig2-deficient mice displayed improved glucose tolerance and reduced liver triglyceride content. Hig2 deficiency increased lipolysis and -oxidation, accounting for the reduced triglyceride accumulation. Similarly, adipocyte-specific Hig2-deficient mice displayed improved glucose tolerance, reduced adipose tissue weight and brown adipose tissue that was largely cleared of lipids. These improvements were abrogated when the animals were placed in thermoneutral housing and brown adipocyte-specific Hig2-deficient mice also displayed improved glucose tolerance, suggesting that active brown fat largely mediates the metabolic phenotype of Hig2 deletion. Thus, this work demonstrates that Hig2 localizes to LDs in liver and adipose tissue and promotes glucose intolerance.

Adipose tissue is one of the most dynamic tissues in the body and is vital for metabolic homeostasis. In the case of excess nutrient uptake, adipose tissue expands to store excess energy in the form of lipids, and in the case of reduced nutrient intake, adipose tissue can shrink and release this energy. Adipocytes are most functional when the balance between these two processes is intact. To understand the molecular mechanisms that drive insulin resistance or conversely preserve the metabolically healthy state in obese individuals, our laboratory performed a screen for differentially regulated adipocyte genes in insulin resistant versus insulin sensitive subjects who had been matched for BMI. From this screen, we identified the type II transmembrane protein tenomodulin (TNMD), which had been previously implicated in glucose tolerance in gene association studies. TNMD was upregulated in omental fat samples isolated from the insulin resistant patient group compared to insulin sensitive individuals. TNMD was predominantly expressed in primary adipocytes compared to the stromal vascular fraction from this adipose tissue. Furthermore, TNMD expression was greatly increased in human preadipocytes by differentiation, and silencing TNMD blocked adipogenic gene induction and adipogenesis, suggesting its role in adipose tissue expansion. Upon high fat diet feeding, transgenic mice overexpressing Tnmd specifically in adipose tissue developed increased epididymal adipose tissue (eWAT) mass without a difference in mean cell size, consistent with elevated in vitro adipogenesis. Moreover, preadipocytes isolated from transgenic epididymal adipose tissue demonstrated higher BrdU incorporation than control littermates, suggesting elevated preadipocyte proliferation. In TNMD overexpressing mice, lipogenic genes PPARG, FASN, SREBP1c and ACLY were upregulated in eWAT as was UCP-1 in brown fat, while liver triglyceride content was reduced. Transgenic animals displayed improved systemic insulin sensitivity, as demonstrated by decreased inflammation and collagen accumulation and increased Akt phosphorylation in eWAT. Thus, the data we present here suggest that TNMD plays a protective role during visceral adipose tissue expansion by promoting adipogenesis and inhibiting inflammation and tissue fibrosis.

In murine and human brown adipose tissue (BAT), mitochondria are powerful generators of heat. Emerging evidence has suggested that the actions of mitochondria extend beyond this conventional biochemical role. In mouse BAT and cultured brown adipocytes, impaired mitochondrial respiratory capacity is accompanied by attenuated expression of Ucp1, a key thermogenic gene, implying a mitochondrial retrograde signaling. However, few have investigated this association in the context of mitochondria-nucleus communication. Using mice with adipose-specific ablation of LRPPRC, a regulator of respiratory capacity, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to transcriptional and metabolic reprogramming of BAT. Impaired respiratory capacity triggers down-regulation of thermogenic and oxidative genes, promoting a storage phenotype in BAT. This retrograde regulation functions by interfering with promoter-specific recruitment of PPARg. In addition, cytosolic calcium may mediate the retrograde signal from mitochondria to nucleus. These data are consistent with a model whereby BAT connects its respiratory capacity to thermogenic gene expression, which in turn contributes to determining its metabolic commitment. Additionally, we find that augmented respiratory capacity activates the thermogenic gene program in inguinal (subcutaneous) white adipose tissue (IWAT) from adipose-specific LRPPRC transgenic mice. When fed a high-fat diet at thermoneutrality, these mice exhibit metabolic improvements as shown by reduced fat mass and improved insulin sensitivity. Furthermore, there is increased recruitment of brown-like adipocytes in IWAT and thus energy expenditure is significantly increased, providing a potential explanation for protection from obesity. These data suggest that augmented respiratory capacity promotes ‘browning’ of IWAT, which has beneficial effects on obesity and diabetes.

<p dir="ltr">Adipose tissue is characterized by the dominant presence of adipocytes, specialized cells adept at lipid metabolism. These adipocytes act as critical nodes, coordinating the complex processes of energy storage and mobilization according to the body's metabolic requirements. Within the adipocyte population of mammals, there are three main subtypes: white, beige, and brown adipocytes. White adipocytes are primarily dedicated to the sequestration of energy in the form of triglycerides. Conversely, beige and brown adipocytes are distinguished by their capacity for thermogenesis, the process of dissipating nutritional energy as heat. The contemporary challenge of chronic overnutrition has precipitated a global surge in obesity and cardiometabolic diseases. Addressing this issue necessitates the maintenance of white adipocyte homeostasis and the enhancement of the quantity and function of thermogenic adipocytes, which are imperative for mitigating the global obesity epidemics.</p><p dir="ltr">Mitochondrion, a multifunctional organelle, is integral to a broad spectrum of cellular processes, including anabolic and catabolic metabolism, bioenergetics, and signal transduction, all of which are essential for maintaining cellular functions and homeostasis. The efficacy of mitochondrial operations is intrinsically linked to their membrane dynamics. In this study, transmission electron microscopy and mass spectrometry were employed to investigate the proteins implicated in the cold-induced mitochondrial membrane remodeling in brown adipocytes. Through this approach, a poorly characterized protein, Family with Sequence Similarity 210 Member A (FAM210A), was identified as a mitochondrial inner membrane protein that is induced by cold stimulation. Subsequent loss-of-function experiments were conducted to elucidate the role of FAM210A in adipocytes. Mice with adipose-specific deletion of <i>Fam210a</i> (<i>Fam210a</i>^<em>AKO</em>) exhibited compromised mitochondrial cristae structure and a reduced thermogenic capacity in brown adipose tissue (BAT), resulting in an increased susceptibility to lethal hypothermia during acute cold challenge. Moreover, in mice with inducible ablation of <i>Fam210a</i> in adipocytes (<i>Fam210</i>^{<em>iAKO</em>}), mitochondrial alterations in BAT were negligible at thermoneutral conditions; however, they exhibited defective cold-induced mitochondrial cristae remodeling, culminating in a progressive loss of cristae and diminished mitochondrial density. Mechanistically, it was determined that FAM210A interacts with mitochondrial protease YME1L and modulates its activity toward OMA1 and OPA1 cleavage, thus compromising cold-induced mitochondrial remodeling in BAT.</p><p dir="ltr">Additionally, this research delved into the role of FAM210A in adipocytes in response to dietary stress by feeding mice with high-fat diet (HFD). The study found a consistent correlation between FAM210A expression and OPA1 cleavage in adipocytes under HFD challenge. Mice lacking FAM210A in all adipocytes and subjected to HFD exhibited lipoatrophy in white adipose tissue (WAT) and a downregulation of genes associated with adipogenesis and lipid metabolism. In contrast, mice with a brown adipocyte-specific ablation of <i>Fam210a </i>(<i>Fam210a</i>^<em>UKO</em>) displayed no significant change in WAT mass but had enlarged livers. Crucially, both <i>Fam210a</i>^<em>AKO</em> and <i>Fam210a</i>^<em>UKO</em> mice presented increased WAT inflammation, deteriorated glucose tolerance, and exacerbated insulin resistance. These findings underscore the pivotal role of FAM210A in brown adipose tissue (BAT) in the preservation of WAT homeostasis and the regulation of systemic glucose clearance in diet-induced obesity.</p><p dir="ltr">In summary, these studies characterize the mitochondrial dynamics in brown adipocytes in response to cold stress, identify a new cold-induced mitochondrial protein, FAM210A, and uncover its functions in adipocytes under cold and dietary stresses. These findings highlight the importance of mitochondrial remodeling in the adaptive response of adipocytes to evolving metabolic demands. This work establishes FAM210A as a key regulator of mitochondrial cristae remodeling, shedding light on the mechanisms that govern mitochondrial plasticity in adipocytes.</p>