Omics approaches for identifying novel molecular targets of metabolic alterations “Modulation of hepatic metabolism and autophagy in MASLD”

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a high prevalent chronic liver disorder driven by ectopic lipid accumulation in hepatocytes and closely associated with obesity, type 2 diabetes, and metabolic syndrome. Current therapeutic approaches mainly target systemic metabolic risk factors and fail to directly address the hepatic molecular mechanisms underlying disease progression, particularly mitochondrial dysfunction, highlighting the need for more targeted therapeutic strategies. Increasing evidence indicates that alterations in mitochondrial homeostasis and autophagy play a central role in MASLD pathogenesis, supporting the need for innovative, mechanism-based therapeutic approaches. Thyroid hormones play a key role in hepatic lipid metabolism and mitochondrial function and therefore represent attractive therapeutic candidates. While 3,5,3'-triiodo-L-thyronine (T3) potently stimulates lipid oxidation and energy expenditure, its clinical use is limited by systemic thyrotoxic effects. In contrast, 3,5-diiodo-L-thyronine (T2), a naturally occurring T3 metabolite, exerts tissue-selective – predominantly hepatic – metabolic effects without disrupting systemic thyroid homeostasis, acting mainly through non-genomic mechanisms that directly modulate mitochondrial function. Autophagy dysfunction further contributes to MASLD pathogenesis by impairing lipid droplet clearance (lipophagy) and removal of damaged mitochondria (mitophagy). Tat-Beclin 1 (Tb), a peptide known as an “autophagy inducer”, represents a pharmacological approach to restore autophagic flux. The combined targeting of mitochondrial metabolism and autophagy may therefore offer complementary therapeutic benefits. This study investigated whether pharmacological modulation of hepatic mitochondrial metabolism and autophagy counteracts MASLD progression. A mouse model of human MASLD was induced by 19 weeks of high-fat diet (HFD, 50% fat). HFD-fed mice were treated short-term (10 days) with the iodothyronines T3 and T2, and Tb, administered alone and in combination with T2. The project was structured around three main aims: (I) to establish and validate the HFD- induced MASLD model and assess the effects of pharmacological interventions on the whole body metabolic phenotype; (II) to characterise hepatic processes, including redox homeostasis, mitochondrial DNA damage and repair mechanisms, and their modulation by pharmacological interventions; (III) to investigate mitochondrial dysfunction-driven inflammatory signalling, mitochondrial quality control mechanisms (MQC - biogenesis, dynamics, and mitophagy), and autophagy, with an in depth analysis of mitochondrial function and electron transport chain composition. After 19 weeks of HFD feeding, mice developed overt MASLD, characterized by dyslipidaemia, insulin resistance, hepatic steatosis, and a hypothyroid state, accompanied by oxidative stress, inflammation, impaired autophagy, disrupted MQC, and defective mitochondrial bioenergetics. Both T3 and T2 showed beneficial effects, reducing adiposity and hepatic lipid accumulation, and improving insulin signalling, fatty acid oxidation, and lipophagy. Moreover, both iodothyronines reduced mtDNA oxidative damage. However, their mechanisms of action differed substantially. T3 stimulated autophagy, mitochondrial biogenesis, and mitophagy, promoting mitochondrial turnover and enhancing respiratory capacity to sustain the hypermetabolic state induced by T3 itself, thereby highlighting limitations to its therapeutic use. The antioxidant, anti-inflammatory, and lipid-lowering effects mediated by T2 were much more pronounced than with the other treatments. In addition, T2 modulated MQC by promoting mitochondrial fusion and mitophagy, and induced mitochondrial respiratory capacity by direct modulation of the electron transport chain. Histological analysis revealed that Tb reduced hepatic steatosis severity by enhancing autophagy- mediated, clearance of lipid droplets without affecting mitochondrial processes, including mitochondrial quality control. Notably, beyond its established role as autophagy inducer, Tb was shown for the first time, to reduce mtDNA oxidative damage by stimulating the base excision repair system, similarly to iodothyronines. The combined administration of Tb and T2 produced an overall beneficial effect on MASLD by combining autophagy-mediated cell clearance with T2-driven metabolic optimisation of mitochondrial function. However, the effects of the combined treatments were not always synergistic, suggesting that simultaneous modulation of autophagy and mitochondrial metabolism may involve complex regulatory interactions. Overall, the reported data identify mitochondrial dysfunction and impaired quality control as a central pathogenic node in MASLD and highlight the therapeutic potential of strategies targeting mitochondrial metabolism and autophagic pathways. Among the pharmacological approaches investigated, T2 emerged as the most effective and well-tolerated intervention, while its combination with an autophagy inducer such as Tb opens new perspectives for multi- target therapeutic strategies in MASLD.