Metadata language
Konopka, Witold (1975- ) : Supervisor
Publisher:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Place of publishing: Date issued/created: Description:[1], 196 pages : illustrations ; 30 cm ; Bibliography ; Summary in English
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 10.10.2025
Type of object: Subject and Keywords:Arcuate nucleus ; c-Fos ; Dicer ; Hyperphagia ; MikroRNA ; Obesity
Abstract:
Energy homeostasis is crucial for maintenance of body weight and is coordinated through the hypothalamus, in particular the arcuate nucleus (ARC) containing the antagonistic neurons responsible for the food intake and appetite control: orexigenic AgRP/NPY, activated during fasting, and anorexigenic POMC/CART, active during satiety. Their activation depends on detection of peripheral satiety signals, as well as intracellularly on gene expression regulated molecules, such as miRNA. The Dicer cKO mice with a neurospecific and tamoxifen-inducible deletion of Dicer1 gene, the key enzyme in the miRNA biogenesis, is characterized by the development of hyperphagic obesity. A mutation in ARC is responsible for the development of the obesity phenotype. The first aim of the study was to determine the metabolic parameters of Dicer cKO mice. A series of experiments were performed to assess the metabolic changes in Dicer cKO mice including the glucose metabolism assessment, as well as the effect of physical activity on the development of obesity and changes in metabolic parameters analysed using metabolic cages. The effect of leptin infusion on food intake and body weight gain was also determined using osmotic pumps. The results indicate sex-dependent transient changes in glucose metabolism during the weight gaining phase. Studies conducted in metabolic cages showed that Dicer cKO mice consume more food and water during the hyperphagic period in both the active and inactive phases of the circadian rhythm, which is in a line with the changes in respiratory exchange ratio. Dicer cKO mice also exhibit reduced energy expenditure. It was also shown that modifications in leptin signaling slightly contribute to the development of obesity in Dicer cKO mice. Deletion of miRNAs may also affect plastic changes in ARC, thus the second aim of the study was to determine the role of the neuroplasticity marker an immediatly early gene c-fos in the development of the obesity phenomenon in Dicer cKO mice. The c-fos plays an important role in the induction of plastic changes during fasting. We performed analysis of c-fos expression in C57Bl/6J mice during fasting in ARC. It was shown that c-fos does not behave like classical IEG, as its expression is chronically elevated after 24 hours of food removal, and c-FosIR cells show co-localization with AgRP/NPY neurons. Thus, the hypothesis was advanced that constant action of peripheral hunger signals exert neuronal excitability in the ARC, preventing the silencing of the c-Fos response. To investigate this phenomenon, an optogenetic tool was used to activate AgRP neurons with the photosensitive channelrhodopsin-2 (ChR2) expression in animals with ad libitum food access. It has been shown that photostimulation of orexigenic neurons leads to immediate food intake and stimulates c-fos expression. However, without peripheral negative energy signals, the c-fos expression is silenced like canonical IEG. The c-fos expression pattern analysed in ARC of Dicer cKO mice showed the elevated levels of c-Fos protein during the hyperphagic period, implying its role in the development of obesity. To determine the role of c-fos in obesity phenotype, the CRISPR/Cas9 system was used to silence its expression. The designed guideRNA sequences were inserted into AAV viral vectors and then injected into ARC of DicerCas9 mice. However, there were no significant differences in body weight and food intake in Dicer cKO mice after administration of viral vectors carring anty-Fos guideRNAs. Instead, it was observed that mutation in orexigenic neurons in Dicer cKO mice led to lack of responsiveness to negative energy status. The obtained results demonstrate the important role of miRNAs in ARC neurons in metabolic control and maintenance of energy homeostasis. Downregulation of miRNAs lead to hyperphagia and obesity, as well as changes in the functioning of neuronal circuits involved in appetite control.
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Copyright holder:Publication made available with the written permission of the author
Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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