Pharmacological modulation of lipid metabolism as a regulator of cellular plasticity, homeostasis, and stress responses in colon and liver cell models

Excess lipid accumulation is a well-established driver of metabolic dysfunction and tissue injury. However, the cellular impact of lipid overload is determined less by the absolute amount of fat than by its qualitative features and intracellular fate. Specifically, metabolic outcomes depend on the type of fatty acids involved and on how they are partitioned within the cell, whether directed toward β-oxidation, incorporated into membrane phospholipids for remodeling, or safely sequestered as neutral lipids within lipid droplets (LDs). When the cell’s lipid-buffering capacity is overwhelmed, maladaptive programs are triggered, converging on endoplasmic reticulum (ER) stress, impaired autophagic flux, redox imbalance, and the activation of regulated cell death pathways. In parallel, bioactive lipid mediators derived from dietary fats, such as oleoylethanolamide (OEA), engage nutrient-sensing networks and can reprogram intracellular lipid trafficking and metabolism, thereby contributing to the restoration of cellular homeostasis. This dissertation aims to determine whether targeted modulation of lipid metabolism, through complementary molecular approaches, can shape cellular plasticity, enhance stress adaptation, and promote survival under lipid overload in hepatic and intestinal cell models. A central objective is to define the underlying metabolic rewiring using proteomics as a systems-level platform to capture global adaptive responses. First, using hepatocyte models, we dissected PA-induced lipotoxicity and identified a critical failure of neutral-lipid buffering linked to Diacylglycerol o-acyltransferase 1 (DGAT1) downregulation, accompanied by ER stress activation and 2 autophagy blockade. Pharmacological activation of Peroxisome Proliferator-Activated Receptor alpha (PPARα) restored adaptive lipid partitioning, rescued DGAT1 expression, improved mitochondrial efficiency, and abolished PA-induced cytotoxicity, supporting PPARα as a mechanistically grounded lever to re-establish lipid homeostasis. Then, we demonstrate that epithelial-mesenchymal transition (EMT) profoundly conditions lipotoxic vulnerability in colorectal cancer cells: epithelial-like cells exhibit limited LDs biogenesis and high sensitivity to saturated fatty acids (i.e. palmitic acid, PA), whereas mesenchymal-like cells display a lipid-buffered phenotype enriched in LDs and enhanced resistance. Here, quantitative proteomics was pivotal in defining the baseline metabolic states and the PA-responsive networks that distinguish these phenotypes, revealing coordinated differences in LDs biogenesis, mitochondrial programs, stress-response modules, and cell-death signaling that would be difficult to capture with targeted assays alone. Finally, an integrated multi-omics workflow was used to delineate OEA metabolism across colon-derived models. This approach combined label-free quantitative proteomics (LC-MS/MS) with rapid SpiderMass lipidomic/metabolomic profiling and time-resolved OEA-d4 uptake kinetics. Proteome-wide analyses uncovered robust but highly context-dependent pathway reorganization, enabling separation of PPARα-aligned components from OEA-specific programs and linking lipid-signature changes to downstream network-level adaptations in trafficking, membrane dynamics, and stress resilience. Collectively, these findings position lipid partitioning as a determinant of cellular fate under lipid-induced stress and nominate DGAT1-dependent LDs buffering and OEA - PPARα signaling as actionable nodes connecting metabolism to plasticity and stress adaptation. Ultimately, proteomics provides the essential framework to resolve state-dependent cell mechanisms.