Resmetirom reduces lipid load, restores THRB expression and prevents cell damage in a human stem cell based in vitro MAFLD model.

Background: Metabolic dysfunction-associated fatty liver disease (MAFLD) distinguishes metabolic-, gene-tic- and environmental disease drivers. In this study, we aim to develop an in vitro model based on human skin-derived precursors (hSKPs) differentiated to hepatic cells (hSKP-HPC) for environmentally driven MAFLD. This model will be used to assess the efficacy of the thyroid hormone receptor beta (THRB)-selective agonist resmetirom (MGL-3196, Madrigal Pharmaceuticals).
Methods: hSKP-HPCs are sequentially exposed for 24h to MAFLD-inducing triggers (fatty acids, fructose and ethanol) followed by an additional 24h-exposure to lipopolysaccharide (LPS) and resmetirom. Then, the cells are fixed with paraformaldehyde and stained with DAPI (nuclei) and BODIPY™ (neutral lipids) for fluorescence microscopy quantification of lipids using ImageJ. Gene expression analysis using RT-qPCR is performed for APOB, CD36, FASN, THRB, SCD and SREBF1. Intracellular ATP levels are measured using the CellTiter Glo® Luminescent Cell Viability Assay.
Results: After 24h, hSKP-HPCs significantly accumulate intracellular lipids. Subsequent exposure to LPS potentiates this effect, along with reduced intracellular ATP content and expression of THRB, which is in line with clinical data. Expression of APOB potently decreases after 48h, indicating diminished lipid export by very low-density lipoproteins (VLDL). Expression of CD36 (fatty acid import) and SREBF1 and its downstream targets SCD and FASN (de novo lipogenesis) also decreases in this condition. Exposure to resmetirom reduces the increased lipid load and restores THRB expression as well as ATP levels back to baseline. Resmetirom also induces the expression of CD36, FASN, SCD and SREBF1, suggesting increased energy turnover by thyroid signaling. In addition, resmetirom potently induces APOB expression, indicating VLDL-mediated triglyceride clearance.
Conclusions: hSKP-HPCs triggered with environmental MAFLD factors exhibit key MAFLD characteristics, including increased lipid load, reduced ATP levels and reduced THRB expression. Resmetirom reduces the lipid load and restores ATP and THRB levels. Therefore, hSKP-HPCs could represent a promising tool for the development of thyromimetic anti-MAFLD drugs.