Desenvolvimento e caracterização molecular de um modelo de ciclagem de peso em peixe-zebra (Danio rerio) e efeitos no metabolismo da descendência.

Obesity is a worldwide epidemic with severe impacts on individuals and society. Although treatments exist, individuals often regain weight loss after a short period in a process called weight cycling. Weight cycling related research is still insufficient, and the murine model used plays a bottleneck for the research in this area. Thus, we aim to develop a novel model of weight cycling in zebrafish, characterize it from a molecular perspective using a multi-omics approach, and elucidate the multigenerational effects of weight cycling in the metabolism of the offspring of weight cycled zebrafish. As a first objective, an experimental protocol using zebrafish to determine the healthy weight and physiological baselines in zebrafish and successfully induce an obesity phenotype in zebrafish using a high-fat diet. For weight cycling, a loss of weight will be induced using a low-fat diet, followed by a regain of weight using a high-fat diet. This objective will establish the weight cycling disease model in zebrafish. The second objective of this research is to validate the experimental procedure by using biometric data such as length, weight, and percentage of fat content based on microcomputed tomography analyses. This will contribute to the robustness of the model and will begin to provide the first insights into noninvasive diagnostic tools for the determination of body fat content in the new model. The third objective consists of determining the metabolomic changes in the blood of zebrafish between experimental groups. This will provide an understanding of the difference between metabolites in healthy, obese, and weight cycles zebrafish. In addition to providing an initial characterization of the model, it also provides insights into new diagnostic methods for weight cycling disease. The fourth objective of this research is to successfully perform an RNAseq analysis and determine the differentially expressed genes related to metabolic pathways in healthy, obese, and weight cycles zebrafish and the development of a Gene Network map, that will allow for the identification of families of metabolic pathways related to each condition tested. This objective will directly address the challenge of finding new molecular pathways targets and genes for the development of better diagnostic and research tools (e.g. genetic tools) as well as targets for drug discovery or therapies. The fifth objective consists of the cross validation of results generated from RNAseq analysis with independent methods for transcriptomic analyses. Most relevant genes identified in RNAseq and gene network analyses will be validated using a quantitative real-time RT-PCR approach. A proteomics approach by western blotting will also be applied here to determine changes at the protein level. The RT-PCR and proteomics validation performed in this step may itself be considered a new genetic-based and protein-based diagnostic tools for direct use in clinical applications. The sixth objective will consist of the determination of the multigenerational effects of weight cycling by analyzing the metabolic responses of the offspring of zebrafish that underwent weight cycling. This will be performed using high throughput assays using zebrafish larvae targeting obesogenic, anti-diabetic, anti-steatosis, and appetite-modulatory activities. This objective will address the lack of knowledge about the possible multigenerational effect that weight cycling might have on our society.