Non-alcoholic fatty liver disease (NAFLD) is a significant global health concern, often associated with obesity, insulin resistance, and inflammation. The liver plays a crucial role in lipid metabolism, balancing lipid uptake, storage, and export. An imbalance between lipid availability and removal via fatty acid oxidation or lipoprotein secretion leads to hepatic lipid accumulation and steatosis. Obesity can overwhelm the liver's capacity to process lipids, contributing to NAFLD progression. Understanding the metabolic regulation of hepatic lipid metabolism is crucial for developing effective therapeutic interventions. While acetyl-CoA plays a central role, the study hypothesized that other metabolic intermediates, such as itaconic acid (itaconate), might also play important, yet undefined roles. Previous research demonstrated itaconate's role in regulating fatty acid β-oxidation, mitochondrial reactive oxygen species (ROS) generation, and metabolic interplay between macrophages and tumors. This study investigated the role of itaconic acid in liver lipid metabolism and NAFLD progression.

The literature review section comprehensively examines existing knowledge on NAFLD pathogenesis, highlighting the liver's central role in lipid homeostasis and the intricate regulatory mechanisms involving hormones, metabolites, and transcription factors. It emphasizes the critical role of acetyl-CoA in lipid metabolism, while suggesting a potential role for other metabolic intermediates. Previous research on itaconate's immunomodulatory properties and its impact on macrophage metabolism is discussed, setting the stage for investigating itaconate's potential influence on hepatic lipid metabolism in the context of NAFLD.

The study employed several methodologies. First, it used unbiased metabolomics to analyze lipid metabolism in bone-marrow macrophages (BMMs) from wild-type and *Irg1*–/– mice, revealing dysregulated lipid metabolism in *Irg1*–/– cells. Next, it examined *Irg1* expression and itaconate production in a mouse model of hyperlipidemia induced by a Western diet (WD), observing upregulation of *Irg1* in hepatic myeloid cells. To determine the cellular source of hepatic itaconate, tissue-specific *Irg1* deletion was employed. Mass spectrometry confirmed itaconate accumulation in F4/80-positive macrophages from WD-fed mice, while hepatocytes showed no detectable itaconate. The study also analyzed human liver biopsies from NASH patients and controls, showing significantly higher *Irg1* mRNA and itaconate levels in NASH livers. To investigate the effects of itaconate, WD-fed mice were treated with 4-octyl itaconate (4-OI), a cell-permeable itaconate derivative. Lipid accumulation was assessed using Oil Red O staining, and metabolic parameters like glucose tolerance, insulin resistance, and serum insulin levels were measured. Finally, primary hepatocytes were treated with itaconate to investigate its direct effects on hepatocyte lipid metabolism and oxidative phosphorylation. Targeted lipidomics, Seahorse flux analysis, and acyl-CoA analysis were used to characterize the metabolic changes induced by itaconate treatment. Statistical analyses (ANOVA, t-tests, Mann-Whitney U-test) were employed to compare groups.

The study found that *Irg1* and itaconate are upregulated in liver macrophages in both a mouse model of NAFLD and human NASH livers. Mice with global or myeloid-specific deletion of *Irg1* exhibited increased adiposity, exacerbated hepatic lipid accumulation (especially triglycerides), glucose intolerance, insulin resistance, and increased mesenteric fat deposition. Treatment of WD-fed *Irg1*–/– mice with 4-OI significantly reduced liver lipid accumulation and mesenteric fat weight. Mechanistically, itaconate treatment of primary hepatocytes decreased lipid accumulation and increased oxidative phosphorylation via fatty acid oxidation. Analysis of macrophages from *Irg1*–/– mice showed accumulation of long-chain fatty acids and reduced levels of metabolites associated with mitochondrial function. Importantly, *Irg1* deficiency primarily in myeloid cells resulted in a phenotype similar to that observed with global *Irg1* deficiency. 4-OI treatment significantly reversed the hyperlipidemic phenotype. The study also found that itaconate treatment of hepatocytes led to increased itaconyl-CoA levels, decreased ATP, and increased ADP levels, suggesting a mechanism of suppressing mitochondrial substrate-level phosphorylation (mSLP). This suppression caused compensatory increases in β-oxidation and glycolysis. In human NASH, increased *Irg1* and itaconate levels were found in the liver tissue. There was a notable reduction in F4/80+ macrophages in both the liver and adipose tissues of *Irg1* deficient mice, suggesting that loss of macrophages might accompany the dysregulated lipid metabolism.

The findings highlight a previously underappreciated role for itaconate in regulating hepatic lipid metabolism. Macrophage-derived itaconate appears to act in *trans* on hepatocytes, enhancing fatty acid oxidation and reducing lipid accumulation. The study's findings in both mouse models and human NASH samples suggest a potential therapeutic role for targeting itaconate or *Irg1* to treat NAFLD. The absence of Nrf2 activation in itaconate-treated hepatocytes might be due to the differences between unmodified itaconate and itaconyl derivatives like 4-OI. The study's data suggest that the increased *Irg1* expression in NAFLD serves as a compensatory mechanism to metabolize excess lipids. The loss of macrophages in *Irg1* deficient mice and the observed metabolic changes suggest a complex interplay between macrophages and hepatocytes in regulating lipid homeostasis during NAFLD.

This research reveals a critical link between immunometabolism and lipid metabolism in the liver, particularly highlighting the role of itaconate in regulating hepatic lipid oxidation. The observed upregulation of *Irg1* and itaconate in both mouse models and human NASH samples emphasizes the potential for therapeutic interventions targeting itaconate levels to manage NAFLD and dyslipidemia. Future research should explore the precise mechanisms of itaconate action in different liver cell types and investigate the potential of itaconate-based therapies for treating NAFLD.

The study primarily used male mice, limiting the generalizability to females. The study focused on the effects of itaconate on hepatocytes and macrophages, and further investigation is needed to explore the potential role of other liver cell types, such as hepatic stellate cells. The use of a cell-permeable itaconate derivative (4-OI) might not perfectly replicate the effects of endogenously produced itaconate.