STUDY OF THE ROLE OF CLASS I HISTONE DEACETYLASES IN DIFFERENTIATION, METABOLISM AND IMMUNOPHENOTYPE OF ADIPOSE TISSUE

INTRODUCTION Epigenome modifications and metabolic dysregulation have been shown to be connected to disease states like obesity and to the associated comorbidities. Epigenome modifiers such as histone deacetylases are involved in the regulation of adipose tissue pathophysiology. However, their specific role in adipocyte differentiation is still a matter of research. Moreover, in obesity excessive accumulation of fat, especially in visceral depots, triggers a low-grade inflammation that is responsible of metabolic dysfunction. The action of immune cells within adipose tissue affects normal metabolic homeostasis. PREVIOUS RESULTS Previous in vitro results from our group showed that inhibition of class I HDACs with MS-275, during early stage of adipocyte differentiation, leads to reduction of lipid droplets size. This phenomenon was accompanied by increased expression of genes regarding adipocyte functionality, lipolysis and fatty acid β-oxidation. However, these events were not observed in terminally differentiated adipocytes. Moreover, in vivo studies showed that HDAC3, a member of class I HDACs, acts as a molecular brake of metabolic rewiring that supports browning in WAT. AIM of this thesis was to further elucidate how class I HDACs are involved in adipocyte differentiation and in determining the metabolic phenotype of pre-adipocytes. Moreover, we aimed to investigate the changes of immune cell populations at different time points of high fat feeding, to fully characterize the phenotypical consequences of Hdac3 ablation in mice. In parallel, we wanted to identify key pathways and early events evoked by Hdac3 gene inactivation through transcriptomic and epigenomic analysis. RESULTS In vitro experiments on adipocytes precursors treated with MS-275 at the beginning of differentiation showed increased expression of genes belonging to mitochondrial activity and browning, which was accompanied by increased H3K27 acetylation of Pparg and Ucp1 gene enhancers. In vivo studies in H3atKO mice, exposed to HFD for different periods (4, 9, 16 weeks, respectively), showed changes in immune cell populations. The number of total macrophages significantly increased in epiWAT of H3atKO mice compared to control floxed (FL) mice at all the three periods of treatment. Moreover, H3atKO mice were able to maintain higher ratio of M2 pro-resolving vs M1 pro-inflammatory macrophages from 4 through 16 weeks of HFD feeding. In H3atKO mice fed LFD for 4 weeks we found upregulation of pathways regarding the futile cycle of simultaneous fatty acid synthesis and β-oxidation. Moreover, KO_LFD vs FL_LFD mice shown upregulation of pathways such as ferroptosis, amino acid biosynthesis and valine, leucine and isoleucine degradation pathways, while focal adhesion and extracellular receptor interaction were downregulated. One of the top downregulated gene was neuronatin (Nnat); we found a hypoacetylated region upstream the Nnat TSS. CONCLUSIONS In vitro results provided evidences regarding the role of class I HDACs in adipocyte differentiation. Their inhibition at the beginning of differentiation promotes an epigenetic imprinting towards oxidative and brown-like phenotype. In vivo experiments revealed that mice lacking Hdac3 were able to maintain a higher ratio of M2 vs M1 macrophages during an inflammatory stimulus such as HFD feeding. Such feature could support a pro-resolving inflammatory process. Omic analysis confirmed the futile cycle of fatty acid metabolism previously observed in our H3atKO model and highlighted interesting new pathways that better characterize H3atKO mice.