Thesis: Jaime Navarro

Directors: Stefania Carobbio

Title: In vitro and in vivo validation and phenotyping of candidate genes uncoupling human adiposity from comorbidities.

Abstract: Obesity is a chronic and heterogeneous disease associated with increased risk of Type 2 diabetes (T2D), cardiovascular diseases (CVDs), metabolic dysfunction-associated steatotic liver disease (MASLD), and cancer. However, not all obese individuals develop complications, highlighting the existence of various metabolic obesity phenotypes. Adipose tissue (AT) expandability and remodelling capacity are key determinants of metabolic health. Healthy AT expansion requires coordinated adipogenesis, membrane lipid remodelling, lipid handling, and extracellular matrix (ECM) dynamics. Impairment of these processes limits AT storage capacity, promoting lipid spillover to peripheral organs and metabolic dysfunction. The molecular mechanisms underlying AT plasticity and the uncoupling of adiposity from metabolic complications are not fully understood. Genome-wide cross-phenotype meta-analyses identified loci associated with increased adiposity but reduced cardiometabolic risk. From these, phospholipase A2 group VI (PLA2G6), ecotropic viral integration site 5 (EVI5), and sarcospan (SSPN) were prioritised. The aim of this thesis is to functionally characterise candidate genes prioritised for their association with the uncoupling of adiposity from metabolic complications. In silico analyses revealed distinct AT expression patterns: PLA2G6 was enriched in mature adipocytes, induced during adipogenesis, preferentially expressed in subcutaneous AT, and inversely associated with obesity status. EVI5 showed broader distribution and early adipogenic regulation, whereas SSPN lacked that consistent patterns, justifying experimental focus on PLA2G6 and EVI5. Stable Evi5 knockdown in 3T3-L1 cells produced no evident phenotype, but transcriptomic profiling revealed modest proadipogenic features, including upregulation of early transcription factors and lipid metabolism genes; and repression of TGF-B signalling, suggesting an early adipogenic role. A double-guide RNA (dgRNA) CRISPR/Cas9 Pla2g6 knockout in 3T3-L1 cells resulted in delayed adipogenesis, impaired mitotic clonal expansion (MCE), reduced adipogenic markers expression, delayed triglyceride (TG) accumulation, and immature ECM remodelling. Lipidomic analyses revealed accumulation of long-chain polyunsaturated fatty acid (PUFA)-enriched glycerophospholipids (GPs) at the sn-2 position, increasing membrane fluidity. Multi-omic integration linked these changes to defective cell cycle progression during MCE, S-phase retention, and p53 accumulation. Pla2g6-KO cells also accumulated free fatty acids (FFAs) and antiadipogenic oxylipins such as prostaglandin E2. Additionally, Pla2g6-KO cells displayed increased mitochondrial number and respiration, reflecting adaptive enhancement of B-oxidation in response to lipid alterations. PPARg agonist rosiglitazone treatment rescued differentiation in Pla2g6-KO cells, promoting hypertrophic adipocytes with larger lipid droplets, although the proliferative defect persisted, indicating uncoupling of MCE from adipogenesis under obesogenic stimulation. In vivo, total Pla2g6-KO mice exhibited a sex-dependent phenotype after 18-weeks high-fat diet (HFD). Males showed preserved body weight (BW) with trends toward increased white AT mass, reduced liver weight, and improved glucose tolerance, consistent with healthier AT expansion. Females displayed reduced BW and food intake and lower adiposity under HFD, and trends toward improved insulin sensitivity. IMPC data suggest bone alterations that could explain those differences. Human genetic data linking lower PLA2G6 expression in AT to adiposity traits and adipocyte size support the translational relevance of these findings. In conclusion, Pla2g6 emerges as a key regulator of AT plasticity through regulation of membrane GP remodelling and fluidity, coordinating adipogenesis and cell cycle progression, mitochondrial adaptation, and metabolically favourable AT expansion; while Evi5 may act in early adipogenic regulation.