The Loss of β-arrestin 2 in Hepatic Macrophages Alleviates Metabolic Dysfunction-associated Steatohepatitis — Through Metabolic Reprogramming of Macrophages

Background and Motivation

Metabolic dysfunction-associated fatty liver disease (MASLD) is a globally prevalent health issue, affecting approximately 25% of the population. This disease includes the milder non-alcoholic fatty liver disease (NAFLD) and the more severe metabolic dysfunction-associated steatohepatitis (MASH). MASH represents the progressive stage of MASLD, characterized by persistent liver damage, inflammation, and fibrosis. With abnormal lipid metabolism, the accumulation of toxic intracellular lipids results in stress-induced damage and death of hepatic cells, thereby triggering an innate immune response and remodeling of the liver microenvironment. Liver macrophages (comprising resident Kupffer cells and monocyte-derived macrophages) are the most abundant innate immune cells in the liver, and their activation plays a critical role in the development of MASH.

Previous studies have pointed out that M1 polarization of macrophages (i.e., pro-inflammatory state) is closely associated with the progression of MASLD, while M2 phenotype macrophages are related to the alleviation of liver damage and improvement of insulin sensitivity. Therefore, regulating the M1/M2 balance of macrophages to control hepatic inflammatory responses could be an effective strategy for treating MASLD. However, the role of macrophage metabolic reprogramming in MASLD and its regulatory mechanisms are still unclear. β-arrestin 2 (ARRB2), as a multifunctional adaptor protein, is closely linked to metabolic disorders, but its regulatory role in immune cell metabolism and MASLD progression remains to be studied. This paper explores the role of ARRB2 in macrophages using various mouse models, revealing the pathogenic mechanism of ARRB2 in MASH.

Research Methods and Process

This study was completed by researchers from several institutions, including Anhui Medical University and Sun Yat-sen University, and published in October 2024 in “Cell Metabolism.” The researchers constructed ARRB2 knockout mice and macrophage-specific ARRB2 knockout models (ARRB2flox/flox Lysozyme-M (Lyz2) Cre mice) to explore the role of ARRB2 in macrophages. They employed various animal models, including high-fat diet (HFD), high-fat high-cholesterol diet (HFHC), and methionine choline-deficient diet (MCD) to induce MASH in mice. To further confirm the impact of ARRB2 on MASLD, the researchers conducted bone marrow transplantation experiments, transplanting ARRB2-KO bone marrow cells into wild-type (WT) mice to observe the effects of the MCD diet on these mice’s liver. Additionally, the research team conducted experiments such as gene set enrichment analysis (GSEA), metabolic pathway analysis (KEGG), hippocampal metabolism analysis, and proteomics to comprehensively evaluate the impact of ARRB2 on macrophage metabolic reprogramming and inflammatory response.

Main Experimental Process

1. Impact of Overall ARRB2 Knockout on Liver Inflammation and Fibrosis: ARRB2-KO mice showed significant weight reduction, decreased liver lipid accumulation, weakened liver inflammatory response, and reduced fibrosis in MASH models induced by HFD, MCD, and HFHC diets. Specific experiments included measurements of liver weight, fat content, ALT and AST levels, and the extent of macrophage and neutrophil infiltration, among other indicators.

2. Protective Effect of Macrophage-specific ARRB2 Knockout on the Liver: Using ARRB2f/f Lyz2Cre mice, researchers further explored the specific role of ARRB2 in macrophages. The ARRB2f/f Lyz2Cre mice showed similar liver protection to ARRB2-KO mice in HFHC and MCD diet models, including weight loss, reduced fat accumulation, lower ALT and AST levels, and reduced fibrosis. Bone marrow transplantation experiments also confirmed the pathogenic role of macrophage ARRB2 in MASH.

3. Effect of ARRB2 on Macrophage M1/M2 Polarization: Flow cytometry revealed a significant reduction in M1 markers and a relative increase in M2 markers in hepatic macrophages of ARRB2-KO mice, suggesting that ARRB2 deletion inhibited the M1 polarization tendency of macrophages. Additionally, gene expression analysis and protein detection showed a significant decrease in the secretion of pro-inflammatory cytokines in ARRB2-KO macrophages, while anti-inflammatory factors like IL-10 increased.

4. Role of ARRB2 in Macrophage Metabolic Reprogramming: ARRB2 deletion inhibited the glycolytic process of macrophages and enhanced mitochondrial oxidative phosphorylation (OxPhos). Experimental results showed that ARRB2 deletion reduced succinate dehydrogenase (SDH) activity, thereby decreasing mitochondrial reactive oxygen species (mtROS) generation, further inhibiting M1 polarization of macrophages.

5. Regulation of the Itaconate Metabolic Pathway by ARRB2: ARRB2 promoted ubiquitination of immune-responsive gene 1 (IRG1), thereby reducing the generation of Itaconate. Itaconate is an endogenous anti-inflammatory metabolite, and the deletion of ARRB2 increased Itaconate levels, thereby inhibiting SDH activity and reducing mtROS production.

Main Findings and Results

1. High Expression of ARRB2 in Hepatic Macrophages of MASLD Patients: The study found that ARRB2 is significantly elevated in hepatic macrophages and peripheral blood monocytes of MASLD patients, and its expression level is positively correlated with the severity of MASLD.

2. ARRB2 Deletion Protects the Liver through Metabolic Reprogramming: ARRB2 deletion promotes the IRG1/Itaconate pathway, inhibits SDH activity, reduces mtROS production, lowers the M1 polarization tendency of macrophages, and ultimately alleviates MASH progression.

3. Metabolic Regulatory Mechanism of ARRB2: ARRB2 promotes ubiquitination and degradation of IRG1, thereby inhibiting Itaconate production. This mechanism reveals ARRB2’s critical role as a multifunctional adaptor protein in the metabolic regulation of macrophages.

4. Clinical Relevance: In MASLD patients, high ARRB2 expression is positively correlated with the degree of hepatic steatosis, ALT and AST levels, suggesting ARRB2 as a potential biomarker for MASLD severity.

Research Significance and Application Value

This study reveals the specific pathogenic role of ARRB2 in macrophages during MASH development, finding that ARRB2 promotes MASH progression through metabolic reprogramming and immune regulation. As a ubiquitination regulator of IRG1, ARRB2 regulates macrophage M1 polarization and inflammatory responses, providing a new potential target for MASH treatment. Furthermore, the positive correlation between ARRB2 expression levels and MASLD severity suggests its potential as a diagnostic marker for MASLD in clinical settings.

Research Highlights and Innovations

1. First clarification of the mechanism by which ARRB2 regulates inflammatory responses through metabolic reprogramming in macrophages, discovering that ARRB2 promotes M1 polarization and inflammatory responses by inhibiting the production of IRG1 and Itaconate.

2. Proposed the potential application of ARRB2 as a diagnostic marker for MASLD, with ARRB2 expression level positively correlating with disease severity, providing a basis for clinical diagnosis.

3. Verified the protective role of ARRB2 deletion in different diet-induced MASH models, including high-fat, high-cholesterol, and methionine and choline-deficient diet models, demonstrating broad applicability.

Limitations and Future Prospects

Although this study reveals the mechanism by which ARRB2 regulates IRG1 and Itaconate metabolism through ubiquitination, confirmation of the specific E3 ubiquitin ligase is lacking. Furthermore, the study primarily relies on mouse models and peripheral blood samples of patients, and has not directly confirmed the regulatory role of ARRB2 on IRG1 metabolic signals in human hepatic macrophages. Moreover, future exploration of the potential application value of macrophage transplantation or gene editing therapies targeting ARRB2 deletion in MASH is warranted.

This research provides new insights into the pathogenesis of MASLD, revealing the critical role of ARRB2 in macrophage metabolism and immune regulation, and holds promise for the development of targeted therapies against ARRB2, offering new therapeutic avenues for MASLD patients.