Pan AMPK-activator O304 ameliorates oxidative stress in MASLD, reverses lipid zonation and reveal lobular metabolic heterogeneitites in the murine live

Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a potential treatment for metabolic dysfunction-associated steatotic liver disease, the most common chronic liver disease worldwide. This study investigates the effect of pan-AMPK activator O304 in a preclinical mouse model of progressive fatty liver disease and demonstrate that O304 reduces body fat mass, blood cholesterol and liver steatosis, as well as ameliorate development of liver fibrosis. These beneficial effects are accompanied by a shift in the metabolic program of the liver including increased lipid oxidation, reduced lipid synthesis, as well as changes in cholesterol and lipid transport. In addition, we detect lobular heterogeneities in the response to O304 and a consistent switch in the zonal distribution of lipid droplets upon treatment. Taken together, our data suggest that O304 is a promising candidate for treatment of MASLD also in human patients 5w old C57Bl6 mice were fed with either standard chow (Special Diet Service #801730) or choline deficient high-fat diet (CD-HFD, D05010402, Research diets). After 3w (short-term cohort) or 21w (long-term cohorts) half of the mice fed CD-HFD were switched to CD-HFD supplemented with 1 mg/g O304 (CD-HFD+O304). Short-term mice were sacrificed 7w after diet-switch while long-term cohorts were sacrificed after 10w or 24w of treatment. Approximately 30 mg of frozen liver were used for total RNA extraction using the RNeasy Plus Universal minikit (Qiagen, #73404). Integrity and quality of RNA and was determined on a Bioanalyzer (Agilent, #5067-1511). Poly(A) RNA was purified with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and RNA-seq libraries was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770) following the manufacturer’s instructions.