Investigating the Effects of Tirzepatide on NAFLD/NASH Progression Using 3D Human Liver Organoids

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is a disease characterized by the ac­cumulation of lipids in the liver that ultimate­ly progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis. Currently, there is no known FDA-approved treatment for NASH. Tirzepatide (brand name Mounjaro), a gluca­gon-like peptide 1 (GLP-1) agonist, has been hypothesized to have potential effects in revers­ing NAFLD/NASH progression and restoring liver function. However, this hypothesis re­mains untested in current literature. The aim of this study is to use 3D human liver organoids (3D-HLOs) as an ex vivo model system to deter­mine the potential effects of tirzepatide on the progression of NAFLD/NASH.

Methods: 3D-HLOs were constructed by in­corporating 5 major human hepatic cell types isolated from NAFLD livers, including hepato­cytes, hepatic stellate cells, liver endothelial cells, cholangiocytes, and Kupffer cells. NAFLD 3D-HLOs were maintained in the hepatocyte medium supplemented with 50mM free fatty acid (FFA) only (control group) or FFA+tirze­patide (100nM, 200nM, or 500 nM) for 14 days. By the end of the treatment, 3D-HLOs were subjected to BODIPY493/503 staining to determine lipid droplet accumulation. Immuno­fluorescence staining and confocal microscopy analysis were performed to confirm hepatocyte function (ALBUMIN) and fibrosis (COL1A1). qPCR was performed to determine the relative expression markers of hepatocyte function (AL­BUMIN, HNF4A), angiogenesis (PECAM-1, VCAM-1, ICAM-1), and fibrosis (ACTA2, COL1A1).

Results: BODIPY 493/503 revealed no sig­nificant difference in lipid deposition between 3D-HLOs in all treatment groups. Mean im­munofluorescence staining intensity for albu­min in controls, 100nM, 200nM, and 500nM tirzepatide were 154.4, 181.4, 221.1, and 236.4, respectively (p>0.05). Dose-dependent qua­dratic regression revealed a strong correlation between dose and albumin fluorescence intensity (R2 = 0.963). qRT-PCR analysis revealed that tirzepatide treatment did not significantly alter transcriptional levels of ALB, HNFA4, ACTA2, PECAM-1, VCAM-1, and ICAM-1 compared to the control group.

Conclusion: Although there is no statistically significant difference in liver cell function mark­ers at different tirzepatide dosages in the setting of NAFLD/NASH, the highest dose of tirzepati­de improved ALB expression despite ongoing FFA exposure, indicating a potential hyperbolic relationship between tirzepatide concentration and albumin production. Future investigation with altered treatment duration and dosage will elucidate whether there are any direct modu­lating effects of tirzepatide on NAFLD/NASH progression.