STUDY ON HISTONE DEACETYLASE 3 AND ITS PHARMACOLOGICAL REGULATION IN THE PATHOPHYSIOLOGY OF ADIPOSE TISSUE

Introduction: Obesity is a chronic complex disease that poses the risk of developing a plethora of serious health complications (e.g., type 2 diabetes, cardiovascular and cerebrovascular disease, and cancers). Browning of white adipose tissue (WAT) can drain excess nutrients via thermogenesis and thus can be used as a strategy to tackle obesity. Epigenetics and metabolism are closely intertwined in both physiological and pathological conditions. Histone deacetylases (HDACs) are epigenome modifiers regulating adipocyte metabolism. Our previous studies with chemical inhibitors or genetic alterations of HDAC3 have shown metabolic rewiring of white adipocytes towards browning via the activation of a futile cycle of lipolysis and lipogenesis. Obesity is characterized by chronic low-grade inflammation due to secretion of pro-inflammatory cytokines by immune cells, but recent studies demonstrated that adipocyte-derived inflammation is necessary for healthy adipose tissue expansion. Ferroptosis is an iron-dependent programmed cell death characterized by the accumulation of lipid peroxides and may have a role in pathophysiology of adipose tissue. Aim: This thesis is focused on the role of histone deacetylase 3 (HDAC3) in WAT browning and obesity with the following aims: 1) assess the effectiveness of HDAC3 knockdown in precursors and mature adipocytes to i) confirm that HDAC3 is the key HDAC that directs adipocyte fate and to ii) evaluate the window of opportunity for this treatment; 2) evaluate the effects of the peculiar metabolic profile observed in HDAC3 KO mice on adipocyte health, viability, and immunophenotype of adipose tissue; 3) study the effects of LA9498, a novel selective inhibitor of HDAC3, in comparison to MS275, a class I HDAC inhibitor which has already been characterized by our group, to evaluate the efficacy and the optimal effective concentration required to achieve browning of white adipocytes. Results: 1) Hdac3 silencing at early stages of differentiation of C3H/10T1/2 adipocytes promoted a metabolic rewiring of the phenotype towards browning. In particular, adipocyte differentiation and functionality, oxidative metabolism, browning, and mitochondrial functionality increased. In addition, adipocyte pro-inflammatory genes were induced by early silencing of Hdac3. On the other hand, silencing Hdac3 in mature adipocytes did not affect cell phenotype. 2) Ablation of HDAC3 in adipocytes determined the increase in total number of macrophages resident in the epiWAT, but this increase was mostly contributed by the M2 subpopulation. Moreover, Hdac3fatKO mice displayed increased proliferation and differentiation of adipocyte progenitors. FACS analysis confirmed increased adipocytes proliferation and macrophage activation following HDAC3 depletion with RNAi in cultured adipocytes. Alongside new adipocytes, we observed dead adipocytes surrounded by macrophages, suggesting WAT renewal. Integrated analysis of omics data and qPCR analysis revealed ferroptosis as one of the upregulated pathways in WAT of Hdac3fatKO mice and in adipocytes upon silencing of HDAC3 with shRNA adenovector; thus, ferroptosis may be a relevant mechanism triggered in Hdac3fatKO mice to remove metabolically exhausted adipocytes. 3) The HDAC3-selective inhibitor LA9498 is more potent than MS275 and causes upregulation of genes important in i) adipocyte differentiation and functionality, ii) lipid mobilization and β-oxidation, and iii) browning and mitochondrial functionality. LDH test did not reveal toxicity of the compounds at the tested concentrations. Furthermore, LA9498 increased lipid accumulation and parameters of mitochondrial functionality (i.e., basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial oxygen consumption). Conclusions: The early epigenetic events mediated by HDAC3 silencing are crucial to commit adipocyte precursors towards a metabolic phenotype characterized by increased oxidative metabolism and mitochondrial function. These results highlight how epigenome modifications of precursor cells could determine the phenotype and functionality of terminally differentiated adipocytes. Immunophenotype of visceral WAT is affected by increased oxidative metabolism of adipocytes in which HDAC3 is deleted. We propose that HDAC3 functions as a metabolic rheostat to modulate WAT rejuvenation by the removal of old adipocytes and differentiation of new progenitors. In addition, specific pharmacological inhibition of HDAC3 with LA9498 induces a transcriptional program similar to Hdac3 silencing, but effects on mitochondrial respiration are boosted; this suggests that the role played by HDAC3 in some cellular processes could be independent from deacetylase activity. Based on the results obtained in these investigations, we propose that regulation of HDAC3 activity could play a crucial role in improving pathophysiology of metabolic disorders in WAT.