# Assessing Nutraceuticals for Hepatic Steatosis: A Standardized In Vitro Approach

## TL;DR

This study evaluates eight nutraceuticals for their ability to reduce liver fat in a standardized lab setting, finding that most do not effectively lower triglycerides.

## Contribution

The paper introduces a standardized in vitro method to quantitatively assess nutraceuticals for liver steatosis.

## Key findings

- Most nutraceuticals tested did not reduce intracellular triglycerides, with only vitamin E showing a reduction.
- Butyrate, berberine, and curcumin unexpectedly increased triglyceride accumulation.
- Fa2N-4 cells were more sensitive to nutraceuticals and drugs than HepG2 cells.

## Abstract

Background/Objectives: Nutraceuticals, including short-chain fatty acids (SCFAs) and antioxidants (AOXs), are nutrient-derived bioactive compounds considered as potential treatments for metabolic-associated steatotic liver disease (MASLD). However, in vitro studies of their effects are limited by inconsistent experimental conditions, including differences in cell lines, methods of steatosis induction, and culture media, and by reliance on qualitative rather than quantitative assessments. Here, we systematically evaluate the anti-steatotic potential of eight commonly used nutraceuticals—three SCFAs (butyrate, acetate, and propionate) and five AOXs (resveratrol, curcumin, berberine, chlorogenic acid, and vitamin E)—using a standardized in vitro approach. Methods: Following a systematic literature review to identify common experimental conditions, we developed an assay to validate steatosis induction and quantified the effects of the nutraceuticals. For our studies we used the HepG2 liver cancer cell line and the Fa2N-4 immortalized hepatocyte cell line. Steatosis was modeled by stimulating cells with free fatty acids and fructose for 48 h. Nutraceuticals were added either concurrently with steatotic stimulation, to assess preventive effects, or after 24 h to assess therapeutic effects. Anti-steatotic drugs (resmetirom, semaglutide, obeticholic acid, and a DGAT2 inhibitor) were included as positive controls. Intracellular triglyceride levels were measured to quantify steatosis. Results: A systematic review of 46 studies revealed large differences in culture conditions, steatosis induction, and nutraceutical assessment. In our experiments, most nutraceuticals did not reduce intracellular triglycerides, with the exception of vitamin E. Surprisingly, butyrate, berberine, and curcumin increased triglyceride accumulation. Resmetirom was the only drug that significantly decreased triglycerides, while obeticholic acid, semaglutide, and the DGAT2 inhibitor showed minimal or inconsistent effects. Fa2N-4 cells were generally more sensitive than HepG2 cells, showing larger absolute changes in triglyceride levels in response to both nutraceuticals and resmetirom. Conclusions: We established a standardized in vitro assay to evaluate the anti-steatotic potential of nutraceuticals. Using this system, we found that SCFAs and AOXs did not consistently reduce intracellular triglycerides, highlighting the need for quantitative assessments and careful validation when studying anti-steatotic interventions in vitro.