Metabolic dysfunction-associated steatotic liver disease (MASLD): insights on pathophysiological mechanisms through metabolomics and fluxomics

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is now estimated that be from 38% of the general population to 70% in subjects with obesity or diabetes. MASLD pathophysiology is associated to diet rich in saturated lipids, sucrose that stimulates de novo lipogenesis (DNL) and accumulation of lipotoxic compounds that can impair cellular metabolism and induces mitochondrial dysfunction. However, if DNL is a major driver of MASLD and a possible target for pharmacological intervention has not yet been clarified. Moreover, whether Western diet impacts mitochondrial function through accumulation of lipotoxic species has been poorly investigated. The goal of my thesis was to study the changes in hepatic lipid metabolism (DNL and lipid profile) induced by MASLD focusing on the de novo synthesis of triglycerides and ceramides measured in vivo using a novel fluxomic method based on deuterium incorporation in newly synthesized lipids after administration water labelled with deuterium. Two animal model of MASLD were studied: mice fed high fat high sucrose (HFHS) diet and methionine choline-deficient (MCD) diet. Both HFHS and MCD diets were associated with hepatic accumulation of lipotoxic species like ceramides, which were more pronounced in MCD. Plasma lipid concentrations were instead increased in mice fed HFHS and decreased in MCD. Both HFHS and MCD induced a change in mitochondrial lipidomic profile with increased phospholipid content, but hepatic mitochondrial function was unaltered, even in presence of hepatic inflammation, indicating that it is not the cause of hepatic fat accumulation and neither of progression of liver disease Among lipids, ceramides are among the ones mostly associated with MASLD as mediators of insulin resistance, thus potentially linked to hepatic steatosis and MASLD progression. Ceramide synthesis and its relationship with DNL and triglyceride (TG) synthesis were studied. Incorporation of fatty acids synthesized by DNL, as palmitate, into hepatic ceramides was increased in HFHS compared to control diets. On the other hand, incorporation of DNL fatty acids in TGs was reduced in HFHS compared to control diets. The effect of weight loss by switching from HFHS diet to a control diet, CC and/or intermittent fasting (IF) was investigated. For this purpose, mice fed with HFHS diet ad libitum were compared with mice that underwent IF without changes diet, switched to CC diet with or without IF, 4 groups. Changes in weight and metabolites/lipids synthesis and concentrations were achieved more in CC+IF group, followed by CC and HFHS+IF group, indicating the importance of diet composition to restore liver metabolism, where caloric restriction is associated with low-fat intake. In conclusion, the results of my thesis have shown that hepatic steatosis, driven by HFHS or MCD diet, does not impair mitochondrial function, although it is associated with lipid remodeling, and is not excluded that in the long run this lipotoxic compounds can cause mitochondrial dysfunction. Although HFHS diet is associated with lower incorporation of newly synthesized (DNL) fatty acids into hepatic triglycerides, I have shown that DNL drives ceramide synthesis, inducing lipotoxicity. Finally, I have shown that not only caloric restriction (by IF), but also diet composition and low-fat intake are important to restore liver metabolism.