Xinru Zhang¹, Charlotte Ehle², Aishwarya Iyer-Bierhoff², Joëlle Wiersema³, Anita Boelen⁴, Eveline Bruinstroop⁵

(1) Endocrine Laboratory, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. (2) Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Jena, Germany. (3) Endocrine Laboratory, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. (4) Endocrine Laboratory, Department of Laboratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. (5) Department of Endocrinology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands. ( 

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum ranging from hepatosteatosis through steatohepatitis to fibrosis and irreversible cirrhosis. Hepatocyte nuclear factor 4A (HNF4A), a nuclear hormone receptor highly expressed in the liver, plays a central role in regulating hepatic lipid metabolism by modulating fat storage via lipophagy. Previously, we identified type 1 iodothyronine deiodinase (DIO1), a key enzyme in thyroid hormone metabolism, as a crucial metabolic regulator in the progression of MASLD. We hypothesized that HNF4A may regulate DIO1 activity, thereby influencing thyroid hormone regulated gene expression in hepatocytes. To test this hypothesis, we examined the effects of HNF4A on DIO1 activity and T3 responsive genes in a HepG2 cell line with an inducible overexpression of HNF4A or HNF4A knock down. 

For HNF4A overexpression, HepG2 cell lines were transfected with a doxycycline-inducible (DOX) myc-tagged HNF4A transgene. For HNF4A knockdown, HepG2 cells were transiently transfected with HNF4A siRNA to reduce HNF4A mRNA expression. DIO1 activity was measured using HPLC with a radioactive rT3 tracer, and DIO1 and T3 responsive gene expression was quantified by RT-qPCR. Cell experiments were at least performed 3 times with a technical triplicate.

Overexpression of HNF4A resulted in a strong increase in DIO1 mRNA expression and DIO1activity compared to control cells. This was accompanied with increased expression of KLF9, G6PC1, SPOT14, CPT1A and PCK1 mRNA. In contrast, knockdown of HNF4A mRNA in HepG2 cells led to a significant decrease in DIO1 expression along with reduced expression of T3 responsive genes.

In conclusion:

This study shows that HNF4A is a potent regulator of DIO1 in hepatocytes. The alterations in DIO1 are associated with contradictory changes in the expression of T3 responsive genes. Further analyses are needed to explain the HNF4A–DIO1 related changes in T3 responsive gene expression but it is clear that HNF4A plays a role in regulating thyroid hormone concentrations via DIO1 in the hepatocyte.