(20b) Stiffness Promotes Metabolic Reprogramming in Primary Hepatocytes

Metabolic-associated fatty liver disease (MAFLD) is the most common liver disease globally and can progress from simple steatosis to nonalcoholic steatohepatitis (NASH) and potentially liver cirrhosis and failure. Hepatocytes are the primary functional liver cell and accumulate excess lipids in MAFLD. Liver stiffness is established as an indicator of disease progression and useful for prognosis. However, the role of liver stiffness in MAFLD and hepatocyte metabolic dysfunction is not well understood. To study the effect of matrix stiffness on hepatocyte function, we cultured primary hepatocytes on our novel biomimetic platform, BEASTS (Bio-Engineered Adhesive Siloxane substrate with Tunable Stiffness). Unlike other common methods for investigating the role of matrix stiffness, such as polyacrylamide gels, BEASTS is able to facilitate cell attachment with a protein-free surface and avoid confounding interactions between the gel’s mechanical properties and adhesive proteins. Using BEASTS, hepatocytes were cultured on a range of stiffnesses representative of healthy (2 kPa) to fibrotic (25, 55 kPa) liver tissue. We observed altered energy metabolism including increased glycolysis in hepatocytes cultured on fibrotic stiffness. Hepatocytes cultured on fibrotic stiffness displayed greater intracellular lipid accumulation with concurrent changes to lipogenic gene expression. We also observed disrupted redox balance in hepatocytes cultured on fibrotic stiffness, with decreased intracellular glutathione and altered inflammatory markers, indicating a pro-inflammatory environment. Our data together implicate increased tissue stiffness in promoting MAFLD-like metabolic changes to hepatocytes in fibrotic liver. This work helps to address a gap in knowledge of how matrix stiffness influences hepatocyte metabolism and to build a more nuanced understanding of disease progression for therapeutic development.