The effect of amino acid-derived metabolites on the development of early metabolic dysfunction-associated steatotic liver disease (MASLD)

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide and its prevalence is rising at an alarming rate given the concurrent rise in its two main risk factors, namely obesity and type 2 diabetes (T2D). In recent years, numerous studies have proposed different endogenous metabolites that are modified in MASLD patients. A collaborative human study identified six metabolites that were altered in MASLD patients receiving resistant starch treatment compared to control MASLD patients. These metabolites have also been correlated with MASLD-related risk factors, yet its mechanism on the development of MASLD is largely unknown. Therefore, this study screened the metabolites in vitro and highlighted its potential role in MASLD development in vivo.

First, the six metabolites were screened by gene expression analysis and valine, 2-aminoadipic acid (AAA) and alpha-aminobutyric acid (ABA) were selected for further analysis as cells pre-treated with these metabolites and then challenged with fatty acids (FA) showed relatively consistent trends in FA uptake and triglyceride synthesis genes that aligned with the human study. RNA sequencing also showed enrichment in several pathways including steroid biosynthesis in both AAA and ABA-treated cells. Analysis on intracellular FA makeup also highlighted greater fold change in stearic acid content in AAA-treated cells while lesser fold change in palmitic and oleic acid content was observed in ABA-treated cells. Therefore, AAA and ABA were selected for subsequent in vivo study.

In the animal study, AAA administration in high-fat diet (HFD)-fed mice consistently reduced hepatic FA oxidation gene expression after 8 and 16 weeks. Relative adipose tissue weight was also significantly higher after 16 weeks of AAA administration despite unchanged average adipocyte size. AAA also elevated hepatic malondialdehyde level significantly after 16 weeks, confirming its role in inducing oxidative stress (OS). Additionally, this study demonstrated the role of AAA in bile acid (BA) metabolism by modifying hepatic BA makeup and consistently suppressing the expression of BA synthesis enzymes namely Cyp7a1 and Cyp27a1 after 8 and 16 weeks.

Conversely, ABA administration in HFD-fed mice exerted minimal effect on hepatic steatosis after 8 weeks. However, after 16 weeks, ABA reduced liver-to-body weight ratio, intrahepatic triglyceride content, and the expression of the FA transporter Cd36 indicating improved hepatic steatosis. Relative adipose tissue weight and adipocyte area were also significantly lower after 16 weeks of ABA administration. ABA also reduced hepatic inflammatory cytokine TNF⍺ level as well as serum ALT and AST level indicating less liver injury. In conclusion, this study outlined the role of AAA in suppressing FA oxidation and confirmed its involvement in OS metabolism. This study also revealed that ABA reduced liver triglyceride by limiting FA uptake and minimised adiposity after 16 weeks. These novel findings provided basis for further investigations on AAA and ABA as potential intervention targets to lessen the occurrence of MASLD.