Identifikation und Charakterisierung des zeitlichen Verlaufs beta-adrenerg kontrollierter Transkripte im braunen und weißen Fettgewebe kältebehandelter Mäuse

Braunes Fettgewebe (BAT) ist bei Kleinsäugern das Organ zur Erzeugung zitterfreier Thermogenese. Die Wärme wird in den spezialisierten Mitochondrien der braunen Adipocyten erzeugt, die das Entkopplerprotein UCP1 enthalten. UCP1 1 fördert die Protonendurchlässigkeit der inneren mitochondrialen Membra...

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Bibliographic Details
Main Author: Schneider, Tatjana
Contributors: Klingenspor, Martin (Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
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Brown and white adipose tissues are innervated by the sympathetic nervous system, which controls metabolic and lipolytic activity in brown and in white adipocytes. However, the metabolic responses and the fate of fatty acids released from intracellular triglycerides by lipolysis are fundamentally different in the two cell types. In animals exposed to cold or overfed with high caloric diets fatty acids are combusted in brown adipocytes to dissipate heat by uncoupling of mitochondrial respiration. In white adipocytes fatty acids released by lipolysis are exported to the circulation and transported to metabolically active peripheral tissues. Morphological and ultrastructural properties of brown and white adipocytes reflect these different functions in thermoregulation and energy balance. Neither in cells differentiated in culture nor in tissues the genes responsible for the functional and morphological divergence between brown and white adipocytes have been studied systematically. Comparison of brown and white preadipocytes has revealed several quantitative differences in gene expression but only a limited number of genes is known to be differentially expressed in brown and white adipocytes. The uncoupling protein-1 (Ucp1) gene is the most prominent representative which is selectively expressed in brown adipocytes and acts as a proton leak in the inner mitochondrial membrane essential for nonshivering thermogenesis in vivo. In cold exposed or overfed rodents increased release of noradrenaline from the sympathetic innervation triggers expresssion of the Ucp1 gene in brown adipocytes. Notably, the genes known to exhibit cell specific expression in brown adipocytes are regulated by β-adrenergic agonists. We therefore performed a systematic search for genes specifically expressed in brown adipocytes in response to isoproterenol stimulation in order to identify new genes which may be required for the thermogenic function of these cells. As brown adipose tissue contains multiple stromal vascular cell types, e.g. preadipocytes, interstitial cells and endothelial cells, a brown adipocyte cell line (HIB-1B) differentiated in culture was chosen for the present screen. In a first step we searched for genes in differentiated HIB 1B brown adipocytes which like Ucp1 display rapid changes in gene expression in response to β-adrenergic stimulation. For this initial screen we applied cDNA representational difference analysis (RDA) comparing brown adipocytes stimulated with isoproterenol with non-stimulated control cells. In a second step differential expression of a random selection of clones from the cDNA subtraction libraries was verified by Northern blotting. Thirdly, 800 cDNA clones obtained by cDNA-RDA were spotted onto microarrays which were then employed to identify true positives in HIB-1B cells and analyse tissue-specific expression of these genes in brown and white adipose tissue. In order to characterize differentially regulated transcripts time course experiments were performed additionally employing cDNA derived from cold exposed mice. We thereby identified genes regulated differentially in brown and white adipose tissue and characterised these transcripts focusing on their time-line expression pattern. Among these were early changes in gene expression profiles of cytoskeleton and tissue modeling proteins indicating intracellular transport activities or structural changes in brown adipose tissue due to adrenergic stimulation. β-adrenerge stimulation triggers differentiation and proliferation of brown adipocytes. Notably we identified genes involved in fatty-acid metabolism including proteins involved in mobilisation and storage of fatty acids. Activation of adaptive thermogenesis during cold exposure is mediated by the adenylyl cyclase pathway. We identified genes in brown and white adipose tissue with different gene expression patterns during the time-course experiment indicating not only differential gene expression among both tissue types but also different responses on transcriptional level. Our experiments lead to the characterisation of an array of genes which to date are completely unknown in brown or white adipose tissue function. Therefore it is likely that activation of adaptive thermogenesis is not coupled to a certain pathway but is linked to many different cellular networks which remain to be elucidated.